Administration of Engineered T Cells for Treatment of Cancers in the Central Nervous System

ABSTRACT

An improved method of treating cancers with engineered T cells is described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of prior co-pending U.S. ProvisionalApplication Ser. No. 62/292,152, filed Feb. 5, 2016, and of priorco-pending U.S. Provisional Application Ser. No. 62/309,348, filed Mar.16, 2016. The disclosures of the above applications are herebyincorporated by reference in their entirety.

BACKGROUND

Tumor-specific T cell based immunotherapies, including therapiesemploying engineered T cells and ex vivo expanded or selected T cells,have been investigated for anti-tumor treatment. In some cases, the Tcells used in such therapies do not remain active in vivo for a longenough period. In some cases, the tumor-specificity of the T cells isrelatively low. In some cases, the engineered T cells have insufficientaccess to the tumor. Therefore, there is a need in the art fortumor-specific cancer therapies with more effective anti-tumor function.

Treatment of cancers of the central nervous system can be particularlychallenging. For example, treatment of high-grade malignant glioma (MG),including anaplastic astrocytoma (AA-grade III) and glioblastomamultiforme (GBM-grade IV), remains a significant therapeutic challenge.Currently available therapeutic options have limited curative potentialand only less than 5% of patients survive more than five years afterinitial diagnosis.

SUMMARY

Described herein are methods for treating malignancies in the centralnervous system by administering compositions comprising T cells (e.g.,CAR T cells, Tumor Infiltrating lymphocytes (“TIL”), TCR-engineered Tcells, or T cell clones) to the cerebrospinal fluid (“CSF”) of apatient. The T cells include T cells that have be manipulated, forexample, by introduction of a nucleic acid molecule expressing a desiredreceptor, by ex vivo expansion of isolated or genetically-modified Tcells or by ex vivo selection of a subset of T cells obtained from apatient or a donor or by a combination of two or more of thesetechniques. Administration to the CNS can be accomplished, for example,by administration to the ventricular system or the central cavity of thespinal column. Administration to the CNS, as the term is used herein, isdistinct from both intratumoral administration (injection or infusioninto the tumor itself) and administration to a cavity created byresection of a tumor. However, the CNS administration methods describedherein can be combined with intraturmoral and/or post-resection,intracavity administration.

The CNS administration described herein permits infusion of relativelylarge volumes of the composition comprising T cells, for example 1 ml-2ml or more in a single infusion. Thus, several million T cells can beadministered in a single infusion.

A method of treating a patient diagnosed with a malignancy of thecentral nervous system is thus disclosed, which comprises infusing acomposition comprising an effective amount of T cells into an anatomicalcompartment of a patient diagnosed with a malignancy of the centralnervous system, the anatomical compartment containing cerebrospinalfluid (“CSF”). The method includes infusion of a composition into aventricular system or a portion of a central canal of a spinal cord, forexample. In one embodiment of the disclosed method, the malignancy ofthe central nervous system includes a primary tumor or a metastasizedtumor found somewhere in the central nervous system, including a portionof the brain, spinal column, or the like. Preferably the anatomicalcompartment contains a contiguous volume of at least about 50, 100, or150 mL of cerebrospinal fluid.

The manipulated T cells infused in the methods described herein targettumor antigens, for example surface protein and intracellular proteins.The malignancies treated can be primary tumors or secondary tumorsarising from cancers originating elsewhere in the body. Becauseadministration to the cerebrospinal fluid allows the T cells access toregions beyond the local site of injection, the methods described hereincan be used to attack and reduce the size of tumors remote from the siteof injection but within the CNS. TCR-engineered T cells are prepared byintroduction of TCRαβ genes into T cells (e.g., autologous T cells)followed by ex vivo expansion of T cells; and infusion of T cells intothe patient. The infusion of the TCR-engineered T cells confers tumorreactivity to patients whose tumor expresses the appropriate antigen andHLA restriction element. The TCR can be targeted to any of a variety oftumor antigens, including, for example melanoma-associated antigenrecognized by T cells 1 (MART-1), glycoprotein (gp) 100,carcinoembryonic antigen (CEA), p53, melanoma-associated antigen(MAGE-A3, and New York esophageal squamous cell carcinoma antigen(NYESO).

Described herein is a method of treating a patient diagnosed with amalignancy of the central nervous system comprising introducing into thecerebrospinal fluid (CSF) of the patient a composition comprising aneffective amount of T cells.

In various embodiments: the T cells are autologous or allogenic T cells;the T cells have been manipulated ex vivo by one or more of: expansion,fractionation or transfection with a recombinant nucleic acid molecule;the T cells comprise cells that have been transfected with a recombinantnucleic acid molecule encoding a polypeptide that binds to a tumor cellantigen; the polypeptide is a chimeric antigen receptor; the compositionis administered intraventricularly; the composition is administered tothe central canal of the spinal cord; the administration is to the leftventrical or the right ventrical; the composition comprises at least1×10⁶ cells; the composition comprising T cells is administered at leasttwo times; the administrations differ in the total number of T cellsadministered; the administrations escalate in dose; the administrationsde-escalate in dose; the T cells comprise CAR T cells; the T cellscomprise autologous tumor infiltrating lymphocytes; the T cells compriseTCR-engineered T cells; the malignancy is a diffuse, infiltrating tumor;the malignancy is a primary brain tumor; one or more tumor foci decreasein size by at least 25%; the malignancy arose from a primary cancerselected from: breast cancer, lung cancer, head and neck cancer, andmelanoma; the method is performed after tumor resection; the methodfurther comprises intratumoral administration of a compositioncomprising T cells; the malignancy is secondary brain tumor; the methodfurther comprises intratumoral administration of a compositioncomprising therapeutic T cells expressing a chimeric antigen receptorthat binds a protein expressed on the surface of glioblastoma cells; thepatient has previously undergone resection of a tumor lesion; the tumorantigen is selected from the group consisting of: IL13Rα2, HER2, PSCA,EGFR, EGFRvIII, EphA2, NY-ESO-1, and CD19; T cells comprise both CD4+cells and CD8+ cells; the T cells have undergone ex vivo expansion; theT cells comprise at least 10% T_(CM) cells; at least 40%, 50%, 60%, 70%or more of the cells infused are CD4+; at least at least 40%, 50%, 60%,70% or more of the cells infused express a cell surface receptor thattargets the tumor antigen (e.g., IL13Rα2); and the dose of cells isbased on the number of infused cells that express a cell surfacereceptor that targets the tumor antigen (e.g., IL13Rα2).

In some embodiments the T cells comprise CART cells that target IL13Rα2and the cells comprise a nucleic acid molecule encoding a chimericantigen receptor comprising: human IL-13 or a variant thereof having1-10 amino acid modifications; a transmembrane domain selected from: aCD4 transmembrane domain or variant thereof having 1-10 amino acidmodifications, a CD8 transmembrane domain or variant thereof having 1-10amino acid modifications, a CD28 transmembrane domain or a variantthereof having 1-10 amino acid modifications, and a CD3ζ transmembranedomain or a variant thereof having 1-10 amino acid modifications; atleast one costimulatory domain; and CD3 signaling domain of a variantthereof having 1-10 amino acid modifications. In some embodiments: thecostimulatory domain is selected from the group consisting of: a CD28costimulatory domain or a variant thereof having 1-10 amino acidmodifications, a 4IBB costimulatory domain or a variant thereof having1-10 amino acid modifications and an OX40 costimulatory domain or avariant thereof having 1-10 amino acid modifications; the variant of ahuman IL13 has 1-10 amino acid modification that increase bindingspecificity for IL13Rα2 versus IL13Rα1; the human IL-13 or variantthereof is an IL-13 variant comprising the amino acid sequence of SEQ IDNO:3 with 1 to 5 amino acid modifications, provided that the amino acidat position 11 of SEQ ID NO:3 is other than E; the chimeric antigenreceptor comprises two different costimulatory domains selected from thegroup consisting of: a CD28 costimulatory domain or a variant thereofhaving 1-10 amino acid modifications, a 4IBB costimulatory domain or avariant thereof having 1-10 amino acid modifications and an OX40costimulatory domain or a variant thereof having 1-10 amino acidmodifications; the chimeric antigen receptor comprises two differentcostimulatory domains selected from the group consisting of: a CD28costimulatory domain or a variant thereof having 1-2 amino acidmodifications, a 4IBB costimulatory domain or a variant thereof having1-2 amino acid modifications and an OX40 costimulatory domain or avariant thereof having 1-2 amino acid modifications; the chimericantigen receptor comprises: human IL-13 or a variant thereof having 1-2amino acid modifications; a transmembrane domain selected from: a CD4transmembrane domain or variant thereof having 1-2 amino acidmodifications, a CD8 transmembrane domain or variant thereof having 1-2amino acid modifications, a CD28 transmembrane domain or a variantthereof having 1-2 amino acid modifications, and a CD3ζ transmembranedomain or a variant thereof having 1-2 amino acid modifications; acostimulatory domain; and CD3 ζ signaling domain of a variant thereofhaving 1-2 amino acid modifications; the CAR comprises a spacer regionlocated between the IL-13 or variant thereof and the transmembranedomain; the spacer region comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO: 4, 14-20, 50 and 52; the chimericantigen receptor comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 10 and 31-48.

In some embodiments the T cells express a chimeric antigen receptor thatbinds HER2 comprise a nucleic acid molecule encoding a chimeric antigenreceptor comprising: a HER2 targeting sequence; a transmembrane domainselected from: a CD4 transmembrane domain or variant thereof having 1-5amino acid modifications, a CD8 transmembrane domain or variant thereofhaving 1-5 amino acid modifications, a CD28 transmembrane domain or avariant thereof having 1-5 amino acid modifications, and a CD3stransmembrane domain or a variant thereof having 1-5 amino acidmodifications; a costimulatory domain selected from a CD28 costimulatorydomain or a variant thereof having 1-5 amino acid modifications and a4-IBB costimulatory domain or a variant thereof having 1-5 amino acidmodifications; and CD3s signaling domain of a variant thereof having 1-5amino acid modifications. In certain embodiments: the HER2 targetingdomain is a HER2 scFv; the HER2 scFv comprising the amino acid sequence:DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO:49)or a variant thereof having 1 to 5 amino acid modifications; thechimeric antigen receptor comprises: a HER2 targeting sequence; atransmembrane domain selected from: a CD4 transmembrane domain orvariant thereof having 1-2 amino acid modifications, a CD8 transmembranedomain or variant thereof having 1-2 amino acid modifications, a CD28transmembrane domain or a variant thereof having 1-2 amino acidmodifications, and a CD3s transmembrane domain or a variant thereofhaving 1-2 amino acid modifications; a costimulatory domain selectedfrom a CD28 costimulatory domain or a variant thereof having 1-2 aminoacid modifications and a 4-IBB costimulatory domain or a variant thereofhaving 1-2 amino acid modifications; and CD3 signaling domain of avariant thereof having 1-2 amino acid modifications; the nucleic acidmolecule expresses a polypeptide comprising an amino acid sequenceselected from SEQ ID NO: 26 and 27 or a variant thereof having 1-5 aminoacid modifications.

Also described herein is a method of treating a patient diagnosed with amalignancy of the central nervous system comprising infusing acomposition comprising an effective amount of T cells into an anatomicalcompartment of a patient diagnosed with a malignancy of the centralnervous system, the anatomical compartment containing cerebrospinalfluid (CSF). In various embodiments: the anatomical compartmentcomprises a portion of a ventricular system; the anatomical compartmentcomprises a portion of a central canal of a spinal cord; the malignancyof the central nervous system includes a brain tumor; the malignancy ofthe central nervous system includes a metastasized tumor; the anatomicalcompartment contains a contiguous volume of at least about 50 mL ofcerebrospinal fluid; the anatomical compartment contains a contiguousvolume of at least about 100 mL of cerebrospinal fluid; and theanatomical compartment contains a contiguous volume of at least about150 mL of cerebrospinal fluid.

Among the cancers that can be treated by the methods described hereinare primary CNS malignancies and secondary malignancies arising from acancer located elsewhere, for example. Acute Lymphoblastic Leukemia(ALL), Acute Myeloid Leukemia (AML), Adrenocortical, Carcinoma,AIDS-Related Cancers, Anal Cancer, Appendix Cancer, Astrocytomas,Atypical Teratoid/Rhabdoid Tumor, Central Nervous System, Basal CellCarcinoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Osteosarcomaand Malignant Fibrous Histiocytoma, Brain Stem Glioma, Brain Tumors,Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumors,Central Nervous System Cancers, Cervical Cancer, Chordoma, ChronicLymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CIVIL),Chronic Myeloproliferative Disorders, Colon Cancer, Colorectal Cancer,Craniopharyngioma, Cutaneous T-Cell Lymphoma, Embryonal Tumors, CentralNervous System, Endometrial Cancer, Ependymoblastoma, Ependymoma,Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma Family of TumorsExtracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor ExtrahepaticBile Duct Cancer, Eye Cancer Fibrous Histiocytoma of Bone, Malignant,and Osteosarcoma, Gallbladder Cancer, Gastric (Stomach) Cancer,Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors(GIST)—see Soft Tissue Sarcoma, Germ Cell Tumor, GestationalTrophoblastic Tumor, Glioma, Hairy Cell Leukemia, Head and Neck Cancer,Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis, HodgkinLymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors(Endocrine Pancreas), Kaposi Sarcoma, Kidney cancer, Langerhans CellHistiocytosis, Laryngeal Cancer, Leukemia, Lip and Oral Cavity Cancer,Liver Cancer (Primary), Lobular Carcinoma In Situ (LCIS), Lung Cancer,Lymphoma, Macroglobulinemia, Male Breast Cancer, Malignant FibrousHistiocytoma of Bone and Osteosarcoma, Medulloblastoma,Medulloepithelioma, Melanoma, Merkel Cell Carcinoma, Mesothelioma,Metastatic Squamous Neck Cancer with Occult Primary Midline TractCarcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine NeoplasiaSyndromes, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides,Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms,Myelogenous Leukemia, Chronic (CML), Myeloid Leukemia, Acute (AML),Myeloma, Multiple, Myeloproliferative Disorders, Nasal Cavity andParanasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, OralCavity Cancer, Oropharyngeal Cancer, Osteosarcoma and Malignant FibrousHistiocytoma of Bone, Ovarian Cancer, Pancreatic Cancer, Papillomatosis,Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, ParathyroidCancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, PinealParenchymal Tumors of Intermediate Differentiation, Pineoblastoma andSupratentorial Primitive Neuroectodermal Tumors, Pituitary Tumor, PlasmaCell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Pregnancy andBreast Cancer, Primary Central Nervous System (CNS) Lymphoma, ProstateCancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis andUreter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma,Salivary Gland Cancer, Sarcoma, Sézary Syndrome, Small Cell Lung Cancer,Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma,Squamous Neck Cancer, Stomach (Gastric) Cancer, Supratentorial PrimitiveNeuroectodermal Tumors, T-Cell Lymphoma, Cutaneous, Testicular Cancer,Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer,Transitional Cell Cancer of the Renal Pelvis and Ureter, TrophoblasticTumor, Ureter and Renal Pelvis Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, WaldenströmMacroglobulinemia, and Wilms Tumor.

In some embodiments, the malignancy treated according to the disclosedmethods comprises a tumor. In some embodiments, treatment results in atleast a 50% reduction in tumor volume, at least a 60% reduction in tumorvolume, at least a 70% reduction in tumor volume, at least an 80%reduction in tumor volume, or at least an 90% reduction in tumor volume.And in some embodiments, the treatment results in elimination of themalignancy.

In some embodiments, the patient does not experience any grade 3 orhigher toxicity.

In some embodiments, the patient was administered a regimen of steroidsprior to treatment with the composition comprising an effective amountof T cells, and in some embodiments the regimen of steroids is reducedto a lower dose following the treatment.

In some embodiments, the patient has an increased life expectancycompared to a patient receiving standard of care treatment, includingradiation therapy, small molecule drug therapy, antibody therapeutics,or a combination thereof. And in some embodiments, in which the patientreceiving standard of care (“SOC”) treatment can expect to survive about15 months from initial diagnosis (overall survival or OS), the patientreceiving the disclosed treatment can expect an OS of 15, 20, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or more. In someembodiments, the patient receiving the claimed treatment can expect anOS of 42, 48, 54, 60, 66, 72, 78, 84, 90 months or more.

In some embodiments, the composition comprises at least 2×10⁶ T cells or2×10⁶ T cells expressing a cell surface receptor targeting a tumorantigen, while in some embodiments, the composition comprises at least1×10⁶ T cells or 1×10⁶ T cells expressing a cell surface receptortargeting a tumor antigen. In some embodiments, the compositioncomprises at least 5×10⁶ T cells or 5×10⁶ T cells expressing a cellsurface receptor targeting a tumor antigen, while in some embodiments,the composition comprises at least 10×10⁶ T cells or 10×10⁶ T cellsexpressing a cell surface receptor targeting a tumor antigen. In someembodiments, the disclosed methods comprised repeated administrations ofthe compositions, for instance repeating administration of thecomposition at least five times, repeating administration of thecomposition at least ten times, or repeating administration until thepatient receives a total dose of at least 90×10⁶ T cells or T cellsexpressing a cell surface receptor targeting a tumor antigen. In someembodiments, the administration is repeated once a week or once everytwo weeks. In some embodiments, the repeated administrations arecontinued over the course of 15 weeks.

Also disclosed herein are methods of increasing a level of at least onecytokine or chemokine in the cerebrospinal fluid (CSF) of a patientcomprising, administering a composition comprising an effective amountof T cells into the CSF of a patient with a malignancy of the centralnervous system, wherein the level of at least one cytokine or chemokinein the CSF is increased following administration of the compositioncomprising an effective amount of T cells compared to a baseline levelof the at least one cytokine or chemokine prior to the administration.

In some embodiments of the disclosed methods, the level of the at leastone cytokine or chemokine in the CSF following the administration isincreased 10-fold or 5-fold compared to the baseline level.

In some embodiments, the level of at least five or at least tencytokines or chemokines is increased following administration of thecomposition comprising an effective amount of T cells compared to abaseline level of the at least five cytokines or chemokines prior to theadministration. In some embodiments, the at least one cytokine orchemokine comprises EGF, Eotaxin, FGF, G-CSF, GM-CSF, HGF, IFN-α, IFN-γ,IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Rα, IL-1β, IL-2, IL-2R, IL-4,IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIG, MIP-1α, MIP-1β, RANTES,TNF-α, or VEGF. In some embodiments, the increase in cytokine orchemokine expression is a local increase (i.e., specific to the CSF).

Also disclosed herein are methods of sustaining for at least about fivedays an increased number of T cells, compared to a baseline number,observed in a cerebrospinal fluid (CSF) of a patient diagnosed with amalignancy of a central nervous system, comprising infusing an effectiveamount of T cells into a CSF of a patient diagnosed with a malignancy ofa central nervous system, in which an increased number of T cellsobserved, compared to a baseline number observed prior to the infusionstep, is sustained for at least about five days.

In some embodiments, an effective amount of T cells (or T cellsexpressing a cell surface receptor that targets the tumor antigen)ranges from about 1×10⁶ cells to about 100×10⁶ cells, and in someembodiments, an effective amount of T cells ranges from about 2×10⁶cells to about 50×10⁶ cells.

In some embodiments, the increased number of T cells observed issustained for at least about six days, or the number of T cells observeddoes not return to the baseline number for about seven days.

In some embodiments, the T cells observed include infused T cells (e.g.CAR-expressing T cells), and in some embodiments, the T cells observedinclude endogenous T cells.

Also disclosed herein are methods of increasing a number of T cells inthe cerebrospinal fluid (CSF) of a patient comprising, administering acomposition comprising an effective amount of T cells into the CSF of apatient with a malignancy of the central nervous system, wherein thenumber of T cells detectable in the CSF is increased compared topre-administration levels.

In some embodiments, the number of T cells detectable in the CSF isincreased compared to pre-administration levels for up to seven daysfollowing administration. In some embodiments, the T cells detectable inthe CSF comprise endogenous T cells and CAR-expressing T cells, and/orType 1 T cells, and/or Type 2 T cells.

In some embodiments, the T cells detectable in the CSF comprise CD3+ Tcells, and in some embodiments, the T cells detectable in the CSFcomprise CD14+ CD11b+ HLA-DR+ mature myeloid populations. In someembodiments, CD19+ B cells and CD11b+ CD15+ granulocytes are detectablein the CSF following administration of the composition.

In some embodiments, reactive lymphocytes, monocytes, and macrophagesare detectable in the CSF following administration of the composition.

Also disclosed herein are methods of determining the suitability of apatient with a malignancy for treatment with an IL-13Rα2-specific CAR Tcell comprising, determining if a score attributed to a sample from thepatient exhibits IL-13Rα2 expression above a predetermined threshold.

In some embodiments, the score attributed to the sample is calculated bydetermining the immunoreactivity of a resected tumor sample from apatient diagnosed with a malignancy by immunohistochemically stainingthe sample with a marker of IL-13Rα2, analyzing the strength of thestaining, and calculating a score based on the strength of the staining,wherein a score that corresponds to moderate to strong stainingintensity in the sample indicates that treatment with anIL-13Rα2-specific CAR T cell is suitable for the patient. In someembodiments, the score comprises counting the number of cells that havea weak, moderate, or strong staining intensity and assigning eachintensity a weight (The H score, a method of quantitatingimmunohistochemical results, is based on the following formula: (3×thepercentage of strongly staining cells)+(2×the percentage of moderatelystaining cells)+(1×the percentage of weakly staining cells), resultingin a range of 0 to 300). In some cases, the patient has a H score thatis: greater that 50, 50-100, greater than 100, 100-200, greater than200, 100-300, or greater than 250 for the relevant tumor-associatedantigen. In some embodiments, an expression of Ki67 in the sample isalso determined by immunohistochemical staining.

Also disclosed herein are methods of treating a patient with amalignancy comprising, administering to a patient diagnosed with amalignancy a composition comprising an effective dose ofIL-13Rα2-specific CART cells, wherein the patient expresses IL-13Rα2above a predetermined threshold.

In some embodiments, the predetermined threshold of IL-13Rα2 expressionwas previously identified as being suitable for a treatment comprisingIL-13Rα2-specific CAR T cell therapy.

TIL are tumor infiltrating lymphocytes that can be isolated from apatient or a donor, expand ex vivo and re-infused into the patient inneed thereof.

CAR T cells express chimeric T cell receptors that comprise anextracellular domain, a transmembrane region and an intracellularsignaling domain. The extracellular domain includes a portion that bindsthe targeted cell and, optionally, a spacer, comprising, for example aportion human Fc domain. The transmembrane portion includes suitabletransmembrane domain, for example, a CD4 transmembrane domain, a CD8transmembrane domain, a CD28 transmembrane domain, a CD3 transmembranedomain or a 4IBB transmembrane domain. The intracellular signalingdomain includes the signaling domain from the zeta chain of the humanCD3 complex (CD3) and one or more costimulatory domains, e.g., a 4-1BBcostimulatory domain. The target cell binding portion of extracellulardomain (for example a scFv or an ligand) enables the CAR, when expressedon the surface of a T cell, to direct T cell activity to those cellsexpressing the targeted cell surface molecule, for example HER2 orIL13Rα2, a receptor expressed on the surface of tumor cells, includingmalignant glioma cells.

A variety of different T cells, for example, patient-specific,autologous T cells, can be engineered to express a TCR or a CAR. VariousT cell subsets can be used. In addition, CAR can be expressed in otherimmune cells such as NK cells. Where a patient is treated with an immunecell expressing a CAR or TCR the cell can be an autologous or allogenicT cell. In some cases, the cells used are CD4+ and CD8+ central memory Tcells (T_(CM)), which are CD45RO+CD62L+, and the use of such cells canimprove long-term persistence of the cells after adoptive transfercompared to the use of other types of patient-specific T cells. TheT_(CM) cells can include CD4+ cells and CD8+ cells.

Among the CAR useful in the methods described herein are those thattarget IL13Rα2. Such CAR can include IL13 having an amino acidmodification, such as an E13Y mutation, that increases bindingspecificity.

The T cells used in the methods described herein can contain a nucleicacid molecule encoding a chimeric antigen receptor (CAR), wherein thechimeric antigen receptor comprises: human IL-13 or a variant thereofhaving 1-10 (e.g., 1 or 2) amino acid modifications; a transmembranedomain selected from: a CD4 transmembrane domain or variant thereofhaving 1-10 (e.g., 1 or 2) amino acid modifications, a CD8 transmembranedomain or variant thereof having 1-10 (e.g., 1 or 2) amino acidmodifications, a CD28 transmembrane domain or a variant thereof having1-10 (e.g., 1 or 2) amino acid modifications, and a CD3ζ transmembranedomain or a variant thereof having 1-10 (e.g., 1 or 2) amino acidmodifications; a costimulatory domain; and CD3 ζ signaling domain of avariant thereof having 1-10 (e.g., 1 or 2) amino acid modifications.

The inclusion of a costimulatory domain, such as the 4-1BB (CD137) orCD28 costimulatory domain in series with CD3ζ in the intracellularregion enables the T cell to receive co-stimulatory signals. Thus, invarious embodiments, the costimulatory domain is selected from the groupconsisting of: a CD28 costimulatory domain or a variant thereof having1-10 (e.g., 1 or 2) amino acid modifications, a 4-IBB costimulatorydomain or a variant thereof having 1-10 (e.g., 1 or 2) amino acidmodifications and an OX40 costimulatory domain or a variant thereofhaving 1-10 (e.g., 1 or 2) amino acid modifications. In certainembodiments, a 4IBB costimulatory domain or a variant thereof having1-10 (e.g., 1 or 2) amino acid modifications is present.

In additional embodiments of the methods, the CAR expressed by the Tcells comprises: a variant of a human IL13 having 1-10 amino acidmodification that increase binding specificity for IL13Rα2 versusIL13Rα1; the human IL-13 or variant thereof is an IL-13 variantcomprising the amino acid sequence of SEQ ID NO:3 with 1 to 5 amino acidmodifications, provided that the amino acid at position 11 of SEQ IDNO:3 other than E; two different costimulatory domains selected from thegroup consisting of: a CD28 costimulatory domain or a variant thereofhaving 1-10 (e.g., 1 or 2) amino acid modifications, a 4IBBcostimulatory domain or a variant thereof having 1-10 (e.g., 1 or 2)amino acid modifications and an OX40 costimulatory domain or a variantthereof having 1-10 (e.g., 1 or 2) amino acid modifications; twodifferent costimulatory domains selected from the group consisting of: aCD28 costimulatory domain or a variant thereof having 1-2 amino acidmodifications, a 4IBB costimulatory domain or a variant thereof having1-2 amino acid modifications and an OX40 costimulatory domain or avariant thereof having 1-2 amino acid modifications; human IL-13 or avariant thereof having 1-2 amino acid modifications; a transmembranedomain selected from: a CD4 transmembrane domain or variant thereofhaving 1-2 amino acid modifications, a CD8 transmembrane domain orvariant thereof having 1-2 amino acid modifications, a CD28transmembrane domain or a variant thereof having 1-2 amino acidmodifications, and a CD3ζ transmembrane domain or a variant thereofhaving 1-2 amino acid modifications; a costimulatory domain; and CD3ζsignaling domain of a variant thereof having 1-2 amino acidmodifications; a spacer region located between the IL-13 or variantthereof and the transmembrane domain (e.g., the spacer region comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:4, 14-20, 50 and 52); the spacer comprises an IgG hinge region; thespacer region comprises 10-150 amino acids; the 4-1BB signaling domaincomprises the amino acid sequence of SEQ ID NO:6; the CD3ζ signalingdomain comprises the amino acid sequence of SEQ ID NO:7; and a linker of3 to 15 amino acids that is located between the costimulatory domain andthe CD3 ζ signaling domain or variant thereof. In certain embodimentswhere there are two costimulatory domains, one is an 4-IBB costimulatorydomain and the other a costimulatory domain selected from: CD28 andCD28gg

In some embodiments of the methods described herein the T cells harbor anucleic acid molecule that expresses a polypeptide comprising an aminoacid sequence selected from SEQ ID NOs: 10 and 31-48; the chimericantigen receptor comprises a IL-13/IgG4/CD4t/41-BB region comprising theamino acid of SEQ ID NO:11 and a CD3 ζ signaling domain comprising theamino acid sequence of SEQ ID NO:7; and the chimeric antigen receptorcomprises the amino acid sequence of SEQ ID NOs: 10 and 31-48.

Also disclosed are methods comprising intraventricular administration ofa population of human T cells transduced by a vector comprising anexpression cassette encoding a chimeric antigen receptor, whereinchimeric antigen receptor comprises: human IL-13 or a variant thereofhaving 1-10 amino acid modifications; a transmembrane domain selectedfrom: a CD4 transmembrane domain or variant thereof having 1-10 aminoacid modifications, a CD8 transmembrane domain or variant thereof having1-10 amino acid modifications, a CD28 transmembrane domain or a variantthereof having 1-10 amino acid modifications, and a CD3ζ transmembranedomain or a variant thereof having 1-10 amino acid modifications; acostimulatory domain; and CD3 signaling domain of a variant thereofhaving 1-10 amino acid modifications. In various embodiments: thepopulation of human T cells comprise a vector expressing a chimericantigen receptor comprising an amino acid sequence selected from SEQ IDNOs: 10 and 31-48; the population of human T cells are comprises ofcentral memory T cells (T_(CM) cells) (e.g., at least 20%, 30%, 40%, 50%60%, 70%, 80% of the cells are T_(CM) cells; at least 10%, 15%, 20%,25%, 30% or 35% of the T cells or the T_(CM) cells are CD4+ and at least10%, 15%, 20%, 25%, 30% or 35% of the T cells or the T_(CM) cells areCD8+ cells).

Also described is a method of treating cancer in a patient comprisingCNS administration of a population of autologous or allogeneic human Tcells (e.g., autologous or allogeneic T cells comprising T_(CM) cells,e.g., at least 20%, 30%, 40%, 50% 60%, 70%, 80% of the cells are T_(CM)cells; at least 15%, 20%, 25%, 30%, 35% of the T_(CM) cells are CD4+ andat least 15%, 20%, 25%, 30%, 35% of the T_(CM) cells are CD8+ cells)transduced by a vector comprising an expression cassette encoding achimeric antigen receptor, wherein chimeric antigen receptor comprisesan amino acid sequence selected from SEQ ID NOs: 10 and 31-48. Invarious embodiments: the population of human T cells comprise centralmemory T cells; the cancer is glioblastoma; and the transduced human Tcells where prepared by a method comprising obtaining T cells from thepatient, treating the T cells to isolate central memory T cells, andtransducing at least a portion of the central memory cells to with aviral vector comprising an expression cassette encoding a chimericantigen receptor, wherein chimeric antigen receptor comprises an aminoacid sequence selected from SEQ ID NOs: 10 and 31-48.

Also described are method is which the T cells administered to thepatient harbor a nucleic acid molecule encoding an polypeptidecomprising an amino acid sequence that is at least 95% identical to anamino acid sequence selected from and SEQ ID NOs: 10 and 31-48; anucleic acid molecule encoding an polypeptide comprising an amino acidsequence that is identical to an amino acid sequence selected from SEQID NO: 10 and 31-48 except for the presence of no more than 5 amino acidsubstitutions, deletions or insertions; a nucleic acid molecule encodingan polypeptide comprising an amino acid sequence that is identical to anamino acid sequence selected from SEQ ID NO:10 and SEQ ID NOs: 10 and31-48 except for the presence of no more than 5 amino acidsubstitutions; and a nucleic acid molecule encoding an polypeptidecomprising an amino acid sequence that is identical to an amino acidsequence selected from SEQ ID NO:10 and SEQ ID NOs: 10 and 31-48 exceptfor the presence of no more than 2 amino acid substitutions.

Described herein are method for treating a patient by CSF administrationof T cells harboring a nucleic acid molecule encoding a chimeric antigenreceptor (CAR), wherein the chimeric antigen receptor comprises an scFvtargeted to HER2 (e.g., comprises the amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS; SEQID NO: 49) or a variant thereof having 1-10 (e.g., 1 or 2) amino acidmodifications; a spacer region; a transmembrane domain selected from: aCD4 transmembrane domain or variant thereof having 1-10 (e.g., 1 or 2)amino acid modifications, a CD8 transmembrane domain or variant thereofhaving 1-10 (e.g., 1 or 2) amino acid modifications, a CD28transmembrane domain or a variant thereof having 1-10 (e.g., 1 or 2)amino acid modifications, and a CD3ζ transmembrane domain or a variantthereof having 1-10 (e.g., 1 or 2) amino acid modifications; acostimulatory domain; and CD3 ζ signaling domain of a variant thereofhaving 1-10 (e.g., 1 or 2) amino acid modifications.

In various embodiments the costimulatory domain is selected from thegroup consisting of: a CD28 costimulatory domain or a variant thereofhaving 1-10 (e.g., 1 or 2) amino acid modifications, a 4-IBBcostimulatory domain or a variant thereof having 1-10 (e.g., 1 or 2)amino acid modifications and an OX40 costimulatory domain or a variantthereof having 1-10 (e.g., 1 or 2) amino acid modifications. In certainembodiments, a 4IBB costimulatory domain or a variant thereof having1-10 (e.g., 1 or 2) amino acid modifications in present.

In additional embodiments T cells administered to the patient express aCAR that comprises: an scFv targeted to HER2 (e.g., a humanized scFv);two different costimulatory domains selected from the group consistingof: a CD28 costimulatory domain or a variant thereof having 1-10 (e.g.,1 or 2) amino acid modifications, a 4IBB costimulatory domain or avariant thereof having 1-10 (e.g., 1 or 2) amino acid modifications andan OX40 costimulatory domain or a variant thereof having 1-10 (e.g., 1or 2) amino acid modifications; two different costimulatory domainsselected from the group consisting of: a CD28 costimulatory domain or avariant thereof having 1-2 amino acid modifications, a 4IBBcostimulatory domain or a variant thereof having 1-2 amino acidmodifications and an OX40 costimulatory domain or a variant thereofhaving 1-2 amino acid modifications; human IL-13 or a variant thereofhaving 1-2 amino acid modifications; a transmembrane domain selectedfrom: a CD4 transmembrane domain or variant thereof having 1-2 aminoacid modifications, a CD8 transmembrane domain or variant thereof having1-2 amino acid modifications, a CD28 transmembrane domain or a variantthereof having 1-2 amino acid modifications, and a CD3ζ transmembranedomain or a variant thereof having 1-2 amino acid modifications; acostimulatory domain; and CD3ζ signaling domain of a variant thereofhaving 1-2 amino acid modifications; a spacer region located between theIL-13 or variant thereof and the transmembrane domain (e.g., the spacerregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 14-20, 50 and 52); the spacer comprises anIgG hinge region; the spacer region comprises 10-150 amino acids; the4-1BB signaling domain comprises the amino acid sequence of SEQ ID NO:6;the CD3ζ signaling domain comprises the amino acid sequence of SEQ IDNO:7; and a linker of 3 to 15 amino acids that is located between thecostimulatory domain and the CD3 ζ signaling domain or variant thereof.In certain embodiments where there are two costimulatory domains, one isan 4-IBB costimulatory domain and the other a costimulatory domainselected from: CD28 and CD28gg

In some embodiments the T cells administered the patient harbor anucleic acid molecule that expresses a polypeptide comprising an aminoacid sequence selected from SEQ ID NOs: 53-56.

Also disclosed are methods comprising intraventricular administration ofa population of human T cells transduced by a vector comprising anexpression cassette encoding a chimeric antigen receptor comprising: anscFv targeted to HER2; a transmembrane domain selected from: a CD4transmembrane domain or variant thereof having 1-10 amino acidmodifications, a CD8 transmembrane domain or variant thereof having 1-10amino acid modifications, a CD28 transmembrane domain or a variantthereof having 1-10 amino acid modifications, and a CD3ζ transmembranedomain or a variant thereof having 1-10 amino acid modifications; acostimulatory domain; and CD3 ζ signaling domain of a variant thereofhaving 1-10 amino acid modifications. In various embodiments: thepopulation of human T cells comprise a vector expressing a chimericantigen receptor comprising an amino acid sequence selected from: SEQ IDNOs: 53-56; the population of human T cells are comprises of centralmemory T cells (T_(CM) cells) (e.g., at least 20%, 30%, 40%, 50% 60%,70%, 80% of the cells are T_(CM) cells; at least 10%, 15%, 20%, 25%,30%, 35% of the T cells or T_(CM) cells are CD4+ and/or at least 10%,15%, 20%, 25%, 30%, 35% of the T cells or T_(CM) cells are CD8+ cells).

Also described is a method of treating cancer in a patient comprisingintraventricular administration of a population of autologous orallogeneic human T cells (e.g., autologous or allogenic T cellscomprising T_(CM) cells, e.g., at least 20%, 30%, 40%, 50% 60%, 70%, 80%of the cells are T_(CM) cells; at least 15%, 20%, 25%, 30%, 35% of theT_(CM) cells are CD4+ and/or at least 15%, 20%, 25%, 30%, 35% of theT_(CM) cells are CD8+ cells) transduced by a vector comprising anexpression cassette encoding a chimeric antigen receptor, whereinchimeric antigen receptor comprises an amino acid sequence selected fromSEQ ID NOs: 53-56. In various embodiments: the population of human Tcells comprise central memory T cells; the cancer is glioblastoma; andthe transduced human T cells where prepared by a method comprisingobtaining T cells from the patient, treating the T cells to isolatecentral memory T cells, and transducing at least a portion of thecentral memory cells to with a viral vector comprising an expressioncassette encoding a chimeric antigen receptor, wherein chimeric antigenreceptor comprises an amino acid sequence selected from SEQ ID NOs:53-56.

Also described is a method of treating cancer in a patient comprisingintraventricular administration of a population of autologous orallogeneic human T cells (e.g., autologous or allogenic T cellscomprising T_(CM) cells harboring: a nucleic acid molecule encoding anpolypeptide comprising an amino acid sequence that is at least 95%identical to an amino acid sequence selected from SEQ ID NOs: 53-56; anucleic acid molecule encoding an polypeptide comprising an amino acidsequence that is identical to an amino acid sequence selected from SEQID NOs: 53-56 except for the presence of no more than 5 amino acidsubstitutions, deletions or insertions; a nucleic acid molecule encodingan polypeptide comprising an amino acid sequence that is identical to anamino acid sequence selected from SEQ ID NOs: 53-56 except for thepresence of no more than 5 amino acid substitutions; and a nucleic acidmolecule encoding an polypeptide comprising an amino acid sequence thatis identical to an amino acid sequence selected from SEQ ID NOs: 53-56,except for the presence of no more than 2 amino acid substitutions.

Certain CAR described herein, for example, the IL13(EQ)BBζ CAR and theIL13(EQ)CD28-BBζ CAR, have certain beneficial characteristics comparedto certain other IL13-targeted CAR. For example, they have improvedselectivity for IL13Rα, elicit lower Th2 cytokine production,particularly lower IL13 production.

T cells expressing a CAR targeting IL13Rα2 can be useful in treatment ofcancers such as glioblastoma, as well as other cancers that expressesIL13Rα2. Thus, this disclosure includes methods for treating cancerusing T cells expressing a CAR described herein.

T cells expressing a CAR targeting HER2 can be useful in treatment ofcancers such as glioblastoma, as well as other cancer that expressesHER2, for example breast cancer that has spread to the central nervoussystem. Thus, this disclosure includes methods for treating cancer usingT cells expressing a CAR described herein.

The CAR described herein can include a spacer region located between thetargeting domain and the transmembrane domain. A variety of differentspacers can be used. Some of them include at least portion of a human Fcregion, for example a hinge portion of a human Fc region or a CH3 domainor variants thereof. Table 1 below provides various spacers that can beused in the CARs described herein.

TABLE 1 Examples of Spacers Name Length Sequence a3 3 aa AAA linker10 aa GGGSSGGGSG (SEQ ID NO: 14) IgG4 hinge (S→P) 12 aaESKYGPPCPPCP (SEQ ID NO: 15) (S228P) IgG4 hinge 12 aaESKYGPPCPSCP (SEQ ID NO: 52) IgG4 hinge + linker 22 aaESKYGPPCPPCPGGGSSGGGSG (SEQ ID NO: 16) CD28 hinge 39 aaIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPS KP (SEQ ID NO: 17) CD8 hinge-48 aa48 aa AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACD (SEQ ID NO: 18)CD8 hinge-45 aa 45 aa TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 19) IgG4(HL-CH3) 129 aaESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 20) IgG4(L235E, N297Q) 229 aaESKYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHQAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4) IgG4(S228P, L235E, N297Q) 229 aaESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHQAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 51) IgG4(CH3) 107 aaGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK (SEQ ID NO: 50)

Some spacer regions include all or part of an immunoglobulin (e.g.,IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that fallsbetween the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fchinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3domain or both a CH3 domain and a CH2 domain. The immunoglobulin derivedsequences can include one ore more amino acid modifications, forexample, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduceoff-target binding.

An “amino acid modification” refers to an amino acid substitution,insertion, and/or deletion in a protein or peptide sequence. An “aminoacid substitution” or “substitution” refers to replacement of an aminoacid at a particular position in a parent peptide or protein sequencewith another amino acid. A substitution can be made to change an aminoacid in the resulting protein in a non-conservative manner (i.e., bychanging the codon from an amino acid belonging to a grouping of aminoacids having a particular size or characteristic to an amino acidbelonging to another grouping) or in a conservative manner (i.e., bychanging the codon from an amino acid belonging to a grouping of aminoacids having a particular size or characteristic to an amino acidbelonging to the same grouping). Such a conservative change generallyleads to less change in the structure and function of the resultingprotein. The following are examples of various groupings of aminoacids: 1) Amino acids with nonpolar R groups: Alanine, Valine, Leucine,Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; 2) Aminoacids with uncharged polar R groups: Glycine, Serine, Threonine,Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with chargedpolar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamicacid; 4) Basic amino acids (positively charged at pH 6.0): Lysine,Arginine, Histidine (at pH 6.0). Another grouping may be those aminoacids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine.

A variety of transmembrane domains can be used in CAR expressed by thecells used in the methods described herein. Table 2 includes examples ofsuitable transmembrane domains. Where a spacer region is present, thetransmembrane domain is located carboxy terminal to the spacer region.

TABLE 2 Examples of Transmembrane Domains Name Accession Length SequenceCD3z J04132.1 21 aa LCYLLDGILFIYGVILTALFL (SEQ ID NO: 21) CD28 NM_00613927 aa FWVLVVVGGVLACYSLLVTVAFII FWV (SEQ ID NO: 22) CD28(M) NM_00613928 aa MFWVLVVVGGVLACYSLLVTVAFI IFWV (SEQ ID NO: 22) CD4 M35160 22 aaMALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 5) CD8tm NM_001768 21 aaIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 23) CD8tm2 NM_001768 23 aaIYIWAPLAGTCGVLLLSLVITLY (SEQ ID NO: 24) CD8tm3 NM_001768 24 aaIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 25) 41BB NM_001561 27 aaIISFFLALTSTALLFLLFF LTLRFSVV (SEQ ID NO: 26)

Many of the CAR expressed by the cells used in the methods describedherein include one or more (e.g., two) costimulatory domains. Thecostimulatory domain(s) are located between the transmembrane domain andthe CD3ζ signaling domain. Table 3 includes examples of suitablecostimulatory domains together with the sequence of the CD3ζ signalingdomain.

TABLE 3 Examples of Costimulatory Domains Name Accession Length SequenceCD3ζ J04132.1 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD28 NM_006139  42 aa RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 27) CD28gg* NM_006139  42 aaRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRS (SEQ ID NO: 28) 41BBNM_001561  42 aa KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 29) OX40  42 aa ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 30)

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic depiction of IL13(E13Y)-zetakine CAR (Left)composed of the IL13Rα2-specific human IL-13 variant (huIL-13(E13Y)),human IgG4 Fc spacer (huγ₄Fc), human CD4 transmembrane (huCD4 tm), andhuman CD3ζ chain cytoplasmic (huCD3ζ cyt) portions as indicated. Alsodepicted is a IL13(EQ)BBζ CAR which is the same as theIL13(E13Y)-zetakine with the exception of the two point mutations, L235Eand N297Q indicated in red, that are located in the CH2 domain of theIgG4 spacer, and the addition of a costimulatory 4-1BB cytoplasmicdomain (4-1BB cyt).

FIGS. 2A-C depict certain vectors and open reading frames. A is adiagram of the cDNA open reading frame of the 2670 nucleotideIL13(EQ)BBZ-T2ACD19t construct, where the IL13Rα2-specific ligandIL13(E13Y), IgG4(EQ) Fc hinge, CD4 transmembrane, 4-1BB cytoplasmicsignaling, three-glycine linker, and CD3 cytoplasmic signaling domainsof the IL13(EQ)BBZ CAR, as well as the T2A ribosome skip and truncatedCD19 sequences are indicated. The human GM-CSF receptor alpha and CD19signal sequences that drive surface expression of the IL13(EQ)BBζ CARand CD19t are also indicated. B is a diagram of the sequences flanked bylong terminal repeats (indicated by ‘R’) that will integrate into thehost genome. C is a map of the IL13(EQ)BBZ-T2A-CD19t_epHIV7 plasmid.

FIG. 3 depicts the construction of pHIV7.

FIG. 4 depicts the elements of pHIV7.

FIG. 5 depicts a production scheme for IL13(EQ)BBζ/CD19t+T_(CM).

FIGS. 6A-C depicts the results of flow cytometric analysis of surfacetransgene and T cell marker expression. IL13(EQ)BBζ/CD19t+T_(CM) HD006.5and HD187.1 were co-stained with anti-IL13-PE and anti-CD8-FITC todetect CD8+ CAR+ and CD4+ (i.e., CD8 negative) CAR+ cells (A), oranti-CD19-PE and anti-CD4-FITC to detect CD4+CD19t+ and CD8+ (i.e., CD4negative) CAR+ cells (B). IL13(EQ)BBζ/CD19t+ T_(CM) HD006.5 and HD187.1stained with fluorochrome conjugated anti-CD3, TCR, CD4, CD8, CD62L andCD28 (grey histograms) or isotype controls (black histograms) (C). Inall cases the percentages based on viable lymphocytes (DAPI negative)stained above isotype.

FIGS. 7A-D depict the results of experiments comparing route of CAR+ Tcell delivery (i.c. versus i.v.) for large established tumors.EGFP-ffLuc+ PBT030-2 TSs (1×10⁵) were implanted into the right forebrainof NSG mice. On days 19 and 26, mice were injected i.v. through the tailvein with either 5×10⁶ CAR+IL13(EQ)BBζ+T_(CM) (11.8×10⁶ total cells;n=4), or mock T_(CM) (11.8×10⁶ cells; n=4). Alternatively, on days 19,22, 26 and 29 mice were injected i.c. with either 1×10⁶CAR+IL13(EQ)BBζ+T_(CM) (2.4×10⁶ total cells; n=4), or mock T_(CM)(2.4×10⁶ cells; n=5). Average ffLuc flux (photons/sec) over time showsthat i.c. delivered IL13(EQ)BBζ T_(CM) mediates tumor regression of day19 tumors. By comparison, i.v. delivered T cells do not shown reductionin tumor burden as compared to untreated or mock T_(CM) controls (A).Kaplan Meier survival curve demonstrates improved survival for micetreated i.c. IL13(EQ)BBZ T_(CM) as compared to mice treated with i.v.administered CAR+T_(CM) (p=0.0003 log rank test) (B). Representative H&Eand CD3 IHC of mice treated i.v. (C) versus i.c. (D) with IL13(EQ)BBZ+T_(CM). CD3+ T cells were only detected in the i.c. treated group, withno CD3+ cells detected in the tumor or surrounding brain parenchyma fori.v. treated mice.

FIGS. 8A-B depict the results of studies showing that CAR+ T cellinjected intracranially, either intratumoral (i.c.t.) orintraventricular (i.c.v.), can traffic to tumors on the oppositehemisphere. EGFP-ffLuc+ PBT030-2 TSs (1×10⁵) were stereotacticallyimplanted into the right and left forebrains of NSG mice. On day 6, micewere injected i.c. at the right tumor site with 1.0×10⁶IL13(EQ)BBζ+T_(CM) (1.6×10⁶ total cells; 63% CAR; n=4). Schematic ofmultifocal glioma experimental model (A). CD3 IHC showing T cellsinfiltrating both the right and left tumor sites (B).

FIG. 9 depicts the amino acid sequence of IL13(EQ)BBζ/CD19t+ (SEQ IDNO:10).

FIG. 10 depicts a sequence comparison of IL13(EQ)41BBζ[IL13{EQ}41BBζT2A-CD19t_epHIV7; pF02630] (SEQ ID NO:12) and CD19Rop_epHIV7 (pJ01683)(SEQ ID NO:13).

FIG. 11 depicts the amino acid sequence of IL13(EmY)-CD8h3-CD8tm2-41BBZeta (SEQ ID NO:31 with GMCSFRa signal peptide; SEQ ID NO:39 withoutGMCSFRa signal peptide).

FIG. 12 depicts the amino acid sequence ofIL13(EmY)-CD8h3-CD28tm-CD28gg-41BB-Zeta (SEQ ID NO:32 with GMCSFRasignal peptide; SEQ ID NO:40 without GMSCFRa signal peptide).

FIG. 13 depicts the amino acid sequence ofIL13(EmY)-IgG4(HL-CH3)-CD4tm-41BB-Zeta (SEQ ID NO:33 with GMCSFRa signalpeptide; SEQ ID NO:41 without GMCSFRa signal peptide).

FIG. 14 depicts the amino acid sequence ofIL13(EmY)-IgG4(L235E,N297Q)-CD8tm-41BB-Zeta (SEQ ID NO:34 with GMCSFRasignal peptide; SEQ ID NO:42 without GMCSFRa signal peptide).

FIG. 15 depicts the amino acid sequence ofIL13(EmY)-Linker-CD28tm-CD28gg-41BB-Zeta (SEQ ID NO:35 with GMCSFRasignal peptide; SEQ ID NO:43 without GMCSFRa signal peptide).

FIG. 16 depicts the amino acid sequence ofIL13(EmY)-HL-CD28m-CD28gg-41BB-Zeta (SEQ ID NO:36 with GMCSFRa signalpeptide; SEQ ID NO:44 without GMSCFRa signal peptide).

FIG. 17 depicts the amino acid sequence ofIL13(EmY)-IgG4(HL-CH3)-CD28tm-CD28gg-41BB-Zeta (SEQ ID NO:37 withGMSCFRa signal peptide; SEQ ID NO:45 without GMCSFRa signal peptide).

FIG. 18 depicts the amino acid sequence of IL13(EmY)IgG4(L235E,N297Q)-CD28tm-CD28gg-41BB-Zeta (SEQ ID NO:38 with GMCSFRasignal peptide; SEQ ID NO:46 without GMCSFRa signal peptide).

FIG. 19 depicts the amino acid sequence of IL13(EmY)-CD8h3-CD8tm-41BBZeta (SEQ ID NO:47 with GMCSFRa signal peptide; SEQ ID NO:48 withoutGMCSFRa signal peptide).

FIG. 20 depicts the amino acid sequence of Her2scFv-IgG4(L235E,N297Q)-CD28tm-CD28gg-Zeta-T2A-CD19t. The various domains are listed inorder below the sequence and are indicated by alternating underliningand non-underlining. The mature CAR sequence (SEQ ID NO:26) does notinclude the GMCSFRa signal peptide, the T2A skip sequence or truncatedCD19.

FIG. 21 depicts the amino acid sequence ofHer2scFv-IgG4(L235E,N297Q)-CD8tm-41BB-Zeta-T2A-CD19t. The variousdomains are listed in order below the sequence and are indicated byalternating underlining and non-underlining. The mature CAR sequence(SEQ ID NO:27) does not include the GMCSFRa signal peptide, the T2A skipsequence or truncated CD19.

FIGS. 22A-D depict HER2-specific CAR constructs and CAR T cell expansiondata.

FIGS. 23A-D depict in vitro characterization of HER2-CAR T cells againstbreast cancer cell lines.

FIGS. 24A-F depict the result of studies on the in vitro tumor activityof HER2-CAR T cells.

FIGS. 25A-I depict the result of studies on the in vivo anti-tumorefficacy of local intratumorally-delivered HER2-CAR T cells.

FIGS. 26A-D depict the results of studies on local delivery of HER2-CART cells in human orthotopic BBM xenograft models.

FIGS. 27A-D depict the results of studies on intraventricular deliveryof HER2-CAR T cells.

FIG. 28 schematically depicts the locations of tumor cell injection andCAR T cell injection for a study of intratumoral and intraventricularinjection of CAR T cells targeting IL13α2R in a murine model ofglioblastoma.

FIGS. 29A-C depict the results of studies demonstrating regression ofestablished glioma tumor xenografts after administration ofIL13(EQ)BBζ/CD19t+ T_(CM). ffLuc⁺ PBT030-2 tumor cells (1×10⁵) werestereotactically implanted into the right and left forebrains of NSGmice. On day 6, mice were injected either ict or icv with 1×10⁶IL13(EQ)BBζ+ Tcm (1.6×10⁶ total cells; 63% CAR+) as described in FIG.4.1 above. A, Representative mice from each group showing relative tumorburden using Xenogen Living Image. B, Average Xenogen flux of left andright brain hemispheres (region of interest, ROI) from the mice (n=4-5)of each group, where each successive bar represents day 5, 9, 12, 15,and 19, respectively. *, p<0.05 when compared to the respective ROI andday/bar of the untreated PBT030-2 group using an unpaired Student'st-test. C, Average Xenogen flux of the whole brain 13 days after T cellinjection. *, p=0.0407 when comparing icv group to untreated PBT030-2group using the unpaired Student's t-test. These data are representativeof three separate multifocal GBM experiments.

FIG. 30 depicts the results of studies demonstrating that huCD3+ cellsare detected in the right and left brain tumors/hemispheres after ictand icv administration of IL13(EQ)BBζ/CD19t+ T_(CM). ffLuc⁺ PBT030-2tumor cells (1×10⁵) were stereotactically implanted into the right andleft forebrains of NSG mice. On day 6, mice were injected either ict(left images) or icv (right images) with 1×10⁶ IL13(EQ)BBζ+ Tcm (1.6×10⁶total cells; 63% CAR+). One week (top images) and two weeks (bottomimages) after T cell administration, 2-3 mice were euthanized from eachgroup, brains were harvested, embedded in paraffin, and IHC wasperformed with anti-human CD3 antibody to detect T cells. RepresentativeIHC images of the left and right tumor sites from mice of each group(ict: m406 and m410; icy: m414 and m415) are depicted. Inlays depict thexenogen flux images of the mice at the day of euthanasia and brainharvest.

FIG. 31 schematically depicts the time course of CAR T cell preparationand treatment for a clinical trial of CAR T cells for treatment ofglioblastoma (A) and provides several dosing schemes (B).

FIG. 32 presents analysis of CAR T cell persistence, as monitored byCD19 (A) and presence of GBM cells as monitored by IL13Rα2 expression(B).

FIG. 33 presents imaging results from Patient UPN097 in the region ofthe catheter used for intratumoral administration.

FIG. 34 is a series of graphs showing the levels of various cytokinesduring the course of treatment for one patient.

FIGS. 35A-C are images depicting egression of recurrent multifocalglioblastoma, including spinal metastasis, following intraventriculardelivery of IL13Rα2-targeted CAR T cells. A patient presenting with arecurrent multifocal GBM, including one metastatic site in the spine andextensive leptomeningeal disease was treated with six local infusions ofIL13BBζ Tcm into the resection cavity of the largest recurrent tumorfocus (1 cycle of 2 M, and 5 cycles of 10M CAR+ T cells). While the CART cell injection site remained stable without evidence of diseaserecurrence for over 7-weeks, other disease foci distant from the CAR Tcell injection site continued to progress (data not shown). This patientthen received five weekly intraventricular (icy) infusions of IL13BBζTcm (1 cycles of 2 M, and 4 cycles of 10M CAR+ T cells). Shown are MRIand/or PET images of (A) transverse brain section, (B) saggital brainsection, and (C) transverse (top) and frontal (bottom) sections of thespine before (left) and one week after (right) completion of i.c.v.therapy, with tumor lesion sites indicated by red arrows in each image.

FIGS. 36A-B shows treatment regimens with enrollment on NCT02208362 anda compassionate use protocol. Enrollment on NCT02208362 was set at day 0(A), with initiation of compassionate use protocol on day 107 (B).NovoTTF-100A, a portable medical device that delivers low intensity,intermediate frequency, alternating electric fields by means ofnoninvasive, disposable scalp electrodes; FGFR, fibroblast growth factorreceptor; MRI, magnetic resonance imaging, all of which was performed onthe brain unless otherwise indicated; ICT, intracavitary; PET, positronemission tomography, performed at the indicated sites; ICV,intracerebroventricular.

FIGS. 37A-C shows immunohistochemistry of primary and recurrent tumors.Tumor resected at initial diagnosis (A), and recurrent tumor (T1)resected at time of Rickham placement under NCT02208362 (B, C).Immunochemical staining using either IL13Rα2-specific or Ki67-specificDAB with hematoxylin counterstain are depicted, with red boxes outliningthe successive magnified images going left to right.

FIGS. 38A-B shows tumor lesion identification. (A) Identifyingcharacteristics of GBM lesions T1-T8. (B) Brain MRI scans depicting thesites of T1-T7.

FIGS. 39A-C shows resected tumor region remains stable, without evidenceof disease progression/recurrence following intracavitary delivery ofIL13BBζ T_(CM). (A) Flow cytometry analysis of the IL13BBζ T_(CM) cellproduct. Top row, transduction with the indicated construct drovecoordinate surface expression of the IL13BBζ CAR (detected withanti-IL13) and the CD19t marker (detected with anti-CD19) via the T2Aribosomal skip sequence. Staining profiles for the T cell markersTCR-α/β, CD3, CD4 and CD8, as well as the exhaustion markers LAG-3,TIM-3, KLRG1 and PD-1 are depicted in the middle row; staining profilesfor the memory markers CD62L, CD45RO, CCR7, CD27, and CD28, as well asthe naïve T cell marker CD45RA are depicted in the bottom row. (B)Treatment schema under NCT02208362. After the patient experiencedrecurrence and underwent tumor excision with placement of anintracavitary (ICT) Rickham catheter, 6 cycles of ICT cell doses (1cycle of 2×10⁶, and 5 cycles of 10×10⁶ CAR+ cells) were administeredwith one week rest between cycles 3 and 4. Red arrow, site of IL13BBζT_(CM) delivery. (C) Yellow circles on brain MM scans show the site ofresected tumor where catheter was placed (T1), as well as theresected-only tumor sites in the frontal lobe (T2, T3), and the newlyarising tumor sites (T6, T7) over the 51-day ICT treatment time period.

FIGS. 40A-E shows regression of recurrent multifocal glioblastoma,including spinal metastases, following intracerebroventricular deliveryof IL13Rα2-targeted CART cells. (A) Treatment schema under compassionateuse protocol. After the patient underwent placement of anintracerebroventricular (ICV) Rickham catheter, 5 cycles of ICV celldoses (1 cycle of 2×10⁶ and 4 cycles of 10×10⁶ CAR+ cells, indicated ascycles 7 through 11) were administered with one week rest between cycles9 and 10. Red arrow, site of IL13BBζ T_(CM) delivery. MM and/or PETimages of (B) transverse brain sections, (C) saggital brain sections,and (D) transverse (top) and frontal (bottom) sections of the spinebefore (left) and one week after (right) completion of ICV therapy, withtumor lesion sites indicated by yellow circles in each image. (E)Maximum lesion areas for non-resected tumors T4-T8 depict theirrespective decreases over time with ICV therapy.

FIG. 41 shows regression of recurrent multifocal GBM following ICVdelivery of IL13Rα2-targeted CAR T cells. Maximum lesion areas fornon-resected tumors T4-T7 are depicted.

FIG. 42A-C shows analysis of cell infiltrates and cytokines fromcerebrospinal fluid (CSF) samples. (A) CSF cellular infiltrate numbersspiked after ICV cycles 9, 10 and 11, with flow cytometric evidence ofCD19+ B cells, both CAR+ (i.e., CD19t+) and non-engineered CD3+ T cells,CD11b+ CD15+ granulocytes, and CD11b+ CD14+ HLA-DR+ monocytes. (B)Evaluation of the CD3+ T cell population in the CSF for the presence ofgene-modified (i.e., CD19t+) T cells. CD3-gated cells from the CSFcollected at the indicated day of cycles 9, 10 and 11 were co-stainedfor CD19 and CD8 (top histograms). Percentages of immunoreactive cellswere then used to calculate numbers of total CD3+ T cells andCD19+CD3+(IL13BBζ T_(CM)) cells per mL of CSF fluid at each time point.(C) Fold change in cytokine levels with ICV treatment cycles 7-11. Onlythose cytokines from the 30-plex analysis that exhibited a 10-fold ormore change compared to pre-treatment levels are depicted.

DETAILED DESCRIPTION

Described below is the structure, construction and characterization ofvarious CAR T cells and their use in treating cancers of the centralnervous system. A chimeric antigen (CAR) is a recombinant biomoleculethat contains, at a minimum, an extracellular recognition domain, atransmembrane region, and an intracellular signaling domain. The term“antigen,” therefore, is not limited to molecules that bind antibodies,but to any molecule that can bind specifically to a target. For example,a CAR can include a ligand that specifically binds a cell surfacereceptor. The extracellular recognition domain (also referred to as theextracellular domain or simply by the recognition element which itcontains) comprises a recognition element that specifically binds to amolecule present on the cell surface of a target cell. The transmembraneregion anchors the CAR in the membrane. The intracellular signalingdomain comprises the signaling domain from the zeta chain of the humanCD3 complex and optionally comprises one or more costimulatory signalingdomains. CARs can both to bind antigen and transduce T cell activation,independent of MHC restriction. Thus, CARs are “universal”immunoreceptors which can treat a population of patients withantigen-positive tumors irrespective of their HLA genotype. Adoptiveimmunotherapy using T lymphocytes that express a tumor-specific CAR canbe a powerful therapeutic strategy for the treatment of cancer.

In some cases the CAR described herein can be produced using a vector inwhich the CAR open reading frame is followed by a T2A ribosome skipsequence and a truncated CD19 (CD19t), which lacks the cytoplasmicsignaling tail (truncated at amino acid 323). In this arrangement,co-expression of CD19t provides an inert, non-immunogenic surface markerthat allows for accurate measurement of gene modified cells, and enablespositive selection of gene-modified cells, as well as efficient celltracking and/or imaging of the therapeutic T cells in vivo followingadoptive transfer. Co-expression of CD19t provides a marker forimmunological targeting of the transduced cells in vivo using clinicallyavailable antibodies and/or immunotoxin reagents to selectively deletethe therapeutic cells, and thereby functioning as a suicide switch.

The disclosed methods of treatment using CAR T cells can be performed atvarious doses and across various timeframes. For example, a patientreceiving an infusion, administration, or injection of CAR T cells (e.g.IL-13Rα2-specific CAR T cells) may receive a single dose comprisingbetween 1×10⁶ and 15×10⁶ cells. In other words, a single dose for use inthe disclosed methods can comprise 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶,6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 10×10⁶, 11×10⁶, 12×10⁶, 13×10⁶, 14×10⁶, or15×10⁶ cells. Over the entire course of treatment, a patient may receivea cumulative or total dose of cells between 20×10⁶ and 150×10⁶ T cells.For instance, the patient may receive about 20×10⁶, about 25×10⁶, about30×10⁶, about 35×10⁶, about 40×10⁶, about 45×10⁶, about 50×10⁶, about55×10⁶, about 60×10⁶, about 65×10⁶, about 70×10⁶, about 75×10⁶, about80×10⁶, about 85×10⁶, about 90×10⁶, about 95×10⁶, about 100×10⁶, about105×10⁶, about 110×10⁶, about 115×10⁶, about 120×10⁶, about 125×10⁶,about 130×10⁶, about 135×10⁶, about 140×10⁶, about 145×10⁶, or about150×10⁶ or more T cells over the course of treatment. In someembodiments, a patient can receive a total dose of at least 90×10⁶ Tcells. In one embodiment, a patient can receive a total dose of 94×10⁶ Tcells.

Furthermore, the doses may be administered according to differentregimens and timetables. For example, the disclosed methods can comprisean infusion, administration, or injection once a day, once every twodays, once every three days, once every four days, once every five days,once every six days, a week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every other month, onceevery three months, or once every six months. In some embodiments, thedisclosed methods can comprise continuous infusion, for instance, from awearable pump. Similarly, the total time course of treatment may beabout 5 weeks, about 10 weeks, about 15 weeks, about 20 weeks, about 25weeks, about 30 weeks, about 35 weeks, about 40 weeks, about 45 weeks,about 50 weeks, about 55 weeks, about 60 weeks, about 65 weeks, about 70weeks, about 75 weeks, or more. The patient may receive 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or moreinfusions, administrations, or injections of T cells over the course oftreatment according to the disclosed methods. For example, in oneembodiment, a patient can receive 11 infusions of T cells over thecourse of 15 weeks.

Treating cancer, and more specifically gliomas like glioblastoma,according to the disclosed methods can result in numerous therapeuticeffects. For instance, treatment with the disclosed CAR T cells canresult in an increase in the level of cytokines and chemokines in theCSF of a patient being treated according to the disclosed methods.Cytokine and/or chemokine expression may increase by at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, or at least 100%,or cytokine and/or chemokine expression may increase by at least 1-fold,at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or atleast 10-fold compared to baseline levels, as measured prior totreatment with a composition comprising CAR T cells. This increase inexpression may be observed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15 or more cytokines or chemokines.

In particular, the expression of at least one of EGF, Eotaxin, FGF,G-CSF, GM-CSF, HGF, IFN-α, IFN-γ, IL-10, IL-12, IL-13, IL-15, IL-17,IL-1Rα, IL-1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1,MIG, MIP-1α, MIP-1β, RANTES, TNF-α, and VEGF may increase as a result oftreatment with CAR T cells as disclosed herein. Furthermore, theincrease in cytokine and/or chemokine expression may be local (i.e. theincrease is only observable in the CNS and CFS, while serum levels ofcytokines and chemokines remain unchanged.

Treatment according to the disclosed methods may also result in anincrease in T cells detectable in the CSF. While at least some of the Tcells detectable in the CSF following treatment will likely beCAR-expressing T cells, there may also be an increase in endogenous Tcells that are recruited to the CSF. Although not wanting to be bound bytheory, the increase in endogenous T cells may be a result of therecruitment of Type 1 and Type 2 T helper cells due to the increase inlocal cytokine levels. Additionally, the detectable T cells can compriseCD3+ T cells, as well as CD14+ CD11b+ HLA-DR+ mature myeloidpopulations, CD19+ B cells and CD11b+CD15+ granulocytes, and/or reactivelymphocytes, monocytes, and macrophages.

The increase in the number of T cells in the CSF may be detectable for aspecific period of time following treatment according to the disclosedmethods. A detectable increase in T cells in the CSF may persist or besustained for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more daysfollowing administration of a composition comprising T cells. Forexample, an increase in the number of T cells observed in the CSF maynot return to baseline levels (i.e. the number of T cells detectableprior to treatment) for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30days. The number of T cells detectable in the CSF may increase by atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 99%, or atleast 100%, or by at least 1-fold, at least 2-fold, at least 3-fold, atleast 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, atleast 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, atleast 12-fold, at least 13-fold, at least 14-fold, or at least 15-foldcompared to baseline levels, as measured prior to treatment with acomposition comprising CAR T cells.

The time course of CAR T cell preparation and treatment is depicted inFIG. 31. Concurrent with the manufacturing process, researchparticipants underwent resection of their tumor(s) followed by placementof a Rickham catheter and baseline imaging.

Patient UPN097 underwent tumor resection and was treated in Cycle 1 with2×10⁶ cells and in Cycle 2 with 10×10⁶ cells. In both Cycle 1 and Cycle2 the cells were administered to the cavity left by resection. After thesecond cycle Patient UPN097 was taken off the study due to rapid tumorprogression.

Patient UPN109 was treated in Cycle 1 with 2×10⁶ cells and in Cycles 2and 3 with 10×10⁶ cells. After a rest period, Patient UPN109 was treatedin Cycles 4, 5 and 6 with 10×10⁶ cells. In Cycles 1-6 the cells wereadministered intratumorally. In Cycle 7 the patient was treated with2×10⁶ cells. In Cycles 8 and 9 the patient was treated with 10×10⁶cells. In Cycles 7-10 the administration was intraventricular.

As used herein, the term “intraventricular” refers to the space insidethe ventricular system, specifically the cerebral ventricles.Accordingly, the term “intraventricular” and “intracerebroventricular”may be used interchangeably throughout this disclosure. Accordingly,“intraventricular administration” or “intraventricular injection” referto delivery of a composition into the ventricals of the brain (i.e. thecerebral ventricles). The cerebral ventricles are a series ofinterconnected, fluid-filled spaces that lie in the core of theforebrain and brainstem. This system comprises four ventricles: theright and left lateral ventricles (one of which is found in eachhemisphere of the brain), the third ventricle, and the fourth ventricle.

The disclosed methods comprise various routes of administering thecompositions comprising T cells. For instance, in some embodiments, thedisclosed compositions may be delivered or administeredintraventricularly. In some embodiments, the disclosed compositions maybe delivered or administered into the spinal canal (i.e. intrathecaldelivery). In some embodiments, the disclosed compositions may bedelivered or administered into the epidural space of the spinal cord(i.e. epidural delivery). In some embodiments, the disclosedcompositions may be delivered or administered directly into a tumor(i.e. intratumoral delivery). In some embodiments, the disclosedcompositions may be delivered or administered into a cavity formed bythe resection of tumor tissue (i.e. intracavity delivery). Furthermore,in some embodiments, the disclosed methods can comprise a combination ofthe aforementioned routes of administration. For instance, a patient mayreceive at least one dose of the composition comprising T cells viaintracavity delivery, followed by at least one dose of the compositionvia intraventricular delivery.

FIG. 32A presents analysis of CAR T cell persistence, as monitored byCD19. This analysis shows good T cell persistence 8 days after the Cycle2. FIG. 32B shows decreased presence of GBM cells as monitored byIL13Rα2 expression.

FIG. 33A and FIG. 33B depict imaging results from Patient UPN097 in theregion of the catheter used for intratumoral administration. In FIG. 33Aone can see that few CD3+ or CD8+ T cells are present pretreatment. FIG.33B, which is a sample at Day 16 post-treatment taken from the leftfrontal tumor cavity wall shows a large area of necrotic tumor andsignificant presence of CD3+ and CD8+ cells.

Gliomas, express IL13 receptors, and in particular, high-affinity IL13receptors. However, unlike the IL13 receptor, glioma cells overexpress aunique IL13Rα2 chain capable of binding IL13 independently of therequirement for IL4Rβ or γc44. Like its homolog IL4, IL13 has pleotropicimmunoregulatory activity outside the CNS. Both IL13 and IL4 stimulateIgE production by B lymphocytes and suppress pro-inflammatory cytokineproduction by macrophages. Detailed studies using autoradiography withradiolabeled IL13 have demonstrated abundant IL13 binding on nearly allmalignant glioma tissues studied. This binding is highly homogeneouswithin tumor sections and in single cell analysis. However, molecularprobe analysis specific for IL13Rα2 mRNA did not detect expression ofthe glioma-specific receptor by normal brain elements andautoradiography with radiolabeled IL13 also could not detect specificIL13 binding in the normal CNS. These studies suggest that the sharedIL13Rα1/IL4β/γc receptor is not expressed detectably in the normal CNS.Therefore, IL13Rα2 is a very specific cell-surface target for glioma andis a suitable target for a CAR designed for treatment of a glioma.

Certain patients may be more suitable than others to receive thedisclosed methods of treatment. For instance, those patients withmalignancies that highly express IL-13Rα2 may particularly benefit fromtreatment with the disclosed CAR T-cells. Suitability of a patient canbe determined by staining a resected tumor sample from a patient todetermine the amount of expression of IL-13Rα2. The sample may be scoredbased on the number of cells exhibiting weak, moderate, or strongstaining intensity. Determining the expression level of Ki67 may also bebeneficial for determining the aggressiveness of the disease. Once ithas been determined that a patient is well suited to receive thedisclosed CAR T cells, the patient may be treated according to thedisclosed methods.

Binding of IL13-based therapeutic molecules to the broadly expressedIL13Rα1/IL4β/γc receptor complex, however, has the potential ofmediating undesired toxicities to normal tissues outside the CNS, andthus limits the systemic administration of these agents. An amino acidsubstitution in the IL13 alpha helix A at amino acid 13 of tyrosine forthe native glutamic acid selectively reduces the affinity of IL13 to theIL13Rα1/IL4β/γc receptor. Binding of this mutant (termed IL13(E13Y)) toIL13Rα2, however, was increased relative to wild-type IL13. Thus, thisminimally altered IL13 analog simultaneously increases IL13'sspecificity and affinity for glioma cells. Therefore, CAR describedherein include an IL13 containing a mutation (E to Y or E to some otheramino acid such as K or R or L or V) at amino acid 13 (according to thenumbering of Debinski et al. 1999 Clin Cancer Res 5:3143s). IL13 havingthe natural sequence also may be used, however, and can be useful,particularly in situations where the modified T cells are to be locallyadministered, such as by injection directly into a tumor mass.

Additionally, gliomas are known to have a generally poor patientprognosis. For example, glioblastoma multiforme (GBM) is a commonmalignant cancer of the CNS. The 1-year and 2-year relative survivalrates for GBM are 29.6% and 9.0%, respectively. Only 3.4% of patientswith a GBM diagnosis survive more than 5 years. Furthermore, recurrencefollowing surgical resection and/or treatment with other conventionaltherapeutics is common. Current conventional treatments include, but arenot limited to, radiation therapy, small molecules (e.g. temozolomide,irinotecan, imatinib mesylate, erlotinib, and hydroxyurea), andbiologics such as antibodies (e.g. bevacizumab).

The disclosed methods of treatment improve clinical prognosis inpatients compared to current standards. For instance, the disclosedmethods can increase 1-year, 2-year, and 5-year survival rates. In someembodiments, the 1-year survival rate of a patient being treatedaccording to the disclosed methods can at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 99%, or at least 100%. In someembodiments, the 2-year survival rate of a patient being treatedaccording to the disclosed methods can at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 99%, or at least 100%. In some embodiments, the5-year survival rate of a patient being treated according to thedisclosed methods can at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 99%, or at least 100%.

In some embodiments, the disclosed methods also increase the lifeexpectancy of a patient compared to another patient receivingconventional treatments or SOC treatment, including radiation therapy,small molecule drug therapy, therapeutic biologics like therapeuticantibodies, or a combination thereof. In some embodiments, in which thepatient receiving SOC treatment can expect to survive about 15 monthsfrom initial diagnosis (overall survival or OS), the patient receivingthe disclosed treatment can expect an OS of 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36 months or more. In some embodiments, the patientreceiving the claimed treatment can expect an OS of 42, 48, 54, 60, 66,72, 78, 84, 90 months or more.

The disclosed methods may improve a patient's prognosis through avariety of clinical outcomes. For instance, the disclosed methods canresult in a reduction in tumor volume in a patient being treated with acomposition comprising T cells. In some embodiments, the disclosedmethods of treatment can result in at least a 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 99%, or at least 100% reduction in tumorvolume. In some embodiments, the tumors in a patient may be completelyeliminated and the patient can be cured of the malignancy.

Additionally, the disclosed methods are safe and well-tolerated.Patients being treated according to the disclosed methods may notexperience significant side effects, and furthermore, may be able todiscontinue taking auxiliary medications. For instance, in someembodiments, the disclosed methods will not result in any grade 3 orhigher toxicities according to NCI Common Toxicity Criteria (CTC). TheCTC provides a quantifiable scale of 0-5, with 0 meaning no adverseevent, 1 meaning mild, 2 meaning moderate, 3 meaning sever andundesirable, 4 meaning life threatening or disabling, and 5 meaningdeath. Thus, side effects and or toxicities may include events like mildor moderate headaches, fatigue, myalgia, and minor nervous systemdisorders such as olfactory aura, but high grade toxicities will beavoided.

Steroids like dexamethasone are commonly used in the clinical managementof gliomas to prevent neurological side effects like brain edema. Thedisclosed methods of treatment can decrease the need for such auxiliarytreatments. For instance, if a patient is receiving a regimen ofsteroids (e.g. dexamethasone) prior to treatment according to thedisclosed methods, the patient may be able to reduce the dose of thesteroid regimen or discontinue the steroid regimen altogether withoutexperiencing clinically deleterious effects.

Brain metastases of breast cancer can express HER2. Certain of the CARdescribed herein that are useful in treatment of malignant glioma aretargeted to HER2.

The CAR described herein can be produced by any means known in the art,though preferably they are produced using recombinant DNA techniques.Nucleic acids encoding the several regions of the chimeric receptor canbe prepared and assembled into a complete coding sequence by standardtechniques of molecular cloning known in the art (genomic libraryscreening, PCR, primer-assisted ligation, site-directed mutagenesis,etc.) as is convenient. The resulting coding region is preferablyinserted into an expression vector and used to transform a suitableexpression host cell line, preferably a T lymphocyte cell line, and mostpreferably an autologous T lymphocyte cell line.

Various T cell subsets isolated from the patient, including unselectedPBMC or enriched CD3 T cells or enriched CD3 or memory T cell subsets,can be transduced with a vector for CAR expression. Central memory Tcells are one useful T cell subset. Central memory T cell can beisolated from peripheral blood mononuclear cells (PBMC) by selecting forCD45RO+/CD62L+ cells, using, for example, the CliniMACS® device toimmunomagnetically select cells expressing the desired receptors. Thecells enriched for central memory T cells can be activated withanti-CD3/CD28, transduced with, for example, a SIN lentiviral vectorthat directs the expression of the CAR as well as a truncated human CD19(CD19t), a non-immunogenic surface marker for both in vivo detection andpotential ex vivo selection. The activated/genetically modified centralmemory T cells can be expanded in vitro with IL-2/IL-15 and thencryopreserved.

Example 1: Construction and Structure of an IL13Rα2-Specific CAR

The structure of a useful IL13Rα2-specific CAR is described below. Thecodon optimized CAR sequence contains a membrane-tethered IL-13 ligandmutated at a single site (E13Y) to reduce potential binding to IL13Rα1,an IgG4 Fc spacer containing two mutations (L235E; N297Q) that greatlyreduce Fc receptor-mediated recognition models, a CD4 transmembranedomain, a costimulatory 4-1BB cytoplasmic signaling domain, and a CD3ζcytoplasmic signaling domain. A T2A ribosome skip sequence separatesthis IL13(EQ)BBζ CAR sequence from CD19t, an inert, non-immunogenic cellsurface detection/selection marker. This T2A linkage results in thecoordinate expression of both IL13(EQ)BBζ and CD19t from a singletranscript. FIG. 1A is a schematic drawing of the 2670 nucleotide openreading frame encoding the IL13(EQ)BBZ-T2ACD19t construct. In thisdrawing, the IL13Rα2-specific ligand IL13(E13Y), IgG4(EQ) Fc, CD4transmembrane, 4-1BB cytoplasmic signaling, three-glycine linker, andCD3ζ cytoplasmic signaling domains of the IL13(EQ)BBZ CAR, as well asthe T2A ribosome skip and truncated CD19 sequences are all indicated.The human GM-CSF receptor alpha and CD19 signal sequences that drivesurface expression of the IL13(EQ)BBZ CAR and CD19t are also indicated.Thus, the IL13(EQ)BBZ-T2ACD19t construct includes a IL13Rα2-specific,hinge-optimized, costimulatory chimeric immunoreceptor sequence(designated IL13(EQ)BBZ), a ribosome-skip T2A sequence, and a CD19tsequence.

The IL13(EQ)BBZ sequence was generated by fusion of the human GM-CSFreceptor alpha leader peptide with IL13(E13Y) ligand 5L235E/N297Q-modified IgG4 Fc hinge (where the double mutation interfereswith FcR recognition), CD4 transmembrane, 4-1BB cytoplasmic signalingdomain, and CD3ζ cytoplasmic signaling domain sequences. This sequencewas synthesized de novo after codon optimization. The T2A sequence wasobtained from digestion of a T2A-containing plasmid. The CD19t sequencewas obtained from that spanning the leader peptide sequence to thetransmembrane components (i.e., basepairs 1-972) of a CD19-containingplasmid. All three fragments, 1) IL13(EQ)BBZ, 2) T2A, and 3) CD19t, werecloned into the multiple cloning site of the epHIV7 lentiviral vector.When transfected into appropriate cells, the vector integrates thesequence depicted schematically in FIG. 1B into the host cells genome.FIG. 1C provides a schematic drawing of the 9515 basepairIL13(EQ)BBZ-T2A-CD19t_epHIV7 plasmid itself.

As shown schematically in FIG. 2, IL13(EQ)BBZ CAR differs in severalimportant respects from a previously described IL13Rα2-specific CARreferred to as IL13(E13Y)-zetakine (Brown et al. 2012 Clinical CancerResearch 18:2199). The IL13(E13Y)-zetakine is composed of theIL13Rα2-specific human IL-13 mutein (huIL-13(E13Y)), human IgG4 Fcspacer (huγ₄Fc), human CD4 transmembrane (huCD4 tm), and human CD3ζchain cytoplasmic (huCD3ζ cyt) portions as indicated. In contrast, theIL13(EQ)BBζ) has two point mutations, L235E and N297Q that are locatedin the CH2 domain of the IgG4 spacer, and a costimulatory 4-1BBcytoplasmic domain (4-1BB cyt).

Example 2: Construction and Structure of epHIV7 Used for Expression ofan IL13Rα2-Specific CAR

The pHIV7 plasmid is the parent plasmid from which the clinical vectorIL13(EQ)BBZ-T2A-CD19t_epHIV7 was derived in the T cell TherapeuticsResearch Laboratory (TCTRL) at City of Hope (COH). The epHIV7 vectorused for expression of the CAR was produced from pHIV7 vector.Importantly, this vector uses the human EF1 promoter to drive expressionof the CAR. Both the 5′ and 3′ sequences of the vector were derived frompv653RSN as previously derived from the HXBc2 provirus. The polypurinetract DNA flap sequences (cPPT) were derived from HIV-1 strain pNL4-3from the NIH AIDS Reagent Repository. The woodchuck post-transcriptionalregulatory element (WPRE) sequence was previously described.

Construction of pHIV7 is schematically depicted in FIG. 3. Briefly,pv653RSN, containing 653 bp from gag-pol plus 5′ and 3′ long-terminalrepeats (LTRs) with an intervening SL3-neomycin phosphotransferase gene(Neo), was subcloned into pBluescript, as follows: In Step 1, thesequences from 5′ LTR to rev-responsive element (RRE) made p5′HIV-1 51,and then the 5′ LTR was modified by removing sequences upstream of theTATA box, and ligated first to a CMV enhancer and then to the SV40origin of replication (p5′HIV-2). In Step 2, after cloning the 3′ LTRinto pBluescript to make p3′HIV-1, a 400-bp deletion in the 3′ LTRenhancer/promoter was made to remove cis-regulatory elements in HIV U3and form p3′HIV-2. In Step 3, fragments isolated from the p5′HIV-3 andp3′HIV-2 were ligated to make pHIV-3. In Step 4, the p3′HIV-2 wasfurther modified by removing extra upstream HIV sequences to generatep3′HIV-3 and a 600-bp BamHI-SalI fragment containing WPRE was added top3′HIV-3 to make the p3′HIV-4. In Step 5, the pHIV-3 RRE was reduced insize by PCR and ligated to a 5′ fragment from pHIV-3 (not shown) and tothe p3′HIV-4, to make pHIV-6. In Step 6, a 190-bp BglII-BamHI fragmentcontaining the cPPT DNA flap sequence from HIV-1 pNL4-3 (55) wasamplified from pNL4-3 and placed between the RRE and the WPRE sequencesin pHIV6 to make pHIV-7. This parent plasmid pHIV7-GFP (GFP, greenfluorescent protein) was used to package the parent vector using afour-plasmid system.

A packaging signal, psi ψ, is required for efficient packaging of viralgenome into the vector. The RRE and WPRE enhance the RNA transcripttransport and expression of the transgene. The flap sequence, incombination with WPRE, has been demonstrated to enhance the transductionefficiency of lentiviral vector in mammalian cells.

The helper functions, required for production of the viral vector), aredivided into three separate plasmids to reduce the probability ofgeneration of replication competent lentivirus via recombination: 1)pCgp encodes the gag/pol protein required for viral vector assembly; 2)pCMV-Rev2 encodes the Rev protein, which acts on the RRE sequence toassist in the transportation of the viral genome for efficientpackaging; and 3) pCMV-G encodes the glycoprotein of thevesiculo-stomatitis virus (VSV), which is required for infectivity ofthe viral vector.

There is minimal DNA sequence homology between the pHIV7 encoded vectorgenome and the helper plasmids. The regions of homology include apackaging signal region of approximately 600 nucleotides, located in thegag/pol sequence of the pCgp helper plasmid; a CMV promoter sequence inall three helper plasmids; and a RRE sequence in the helper plasmidpCgp. It is highly improbable that replication competent recombinantvirus could be generated due to the homology in these regions, as itwould require multiple recombination events. Additionally, any resultingrecombinants would be missing the functional LTR and tat sequencesrequired for lentiviral replication.

The CMV promoter was replaced by the EF1α-HTLV promoter (EF1p), and thenew plasmid was named epHIV7 (FIG. 4). The EF1p has 563 bp and wasintroduced into epHIV7 using NruI and NheI, after the CMV promoter wasexcised.

The lentiviral genome, excluding gag/pol and rev that are necessary forthe pathogenicity of the wild-type virus and are required for productiveinfection of target cells, has been removed from this system. Inaddition, the IL13(EQ)BBZ-T2ACD19t_epHIV7 vector construct does notcontain an intact 3′LTR promoter, so the resulting expressed and reversetranscribed DNA proviral genome in targeted cells will have inactiveLTRs. As a result of this design, no HIV-I derived sequences will betranscribed from the provirus and only the therapeutic sequences will beexpressed from their respective promoters. The removal of the LTRpromoter activity in the SIN vector is expected to significantly reducethe possibility of unintentional activation of host genes. Table 4summarizes the various regulator elements present inIL13(EQ)BBZ-T2ACD19t_epHIV7.

TABLE 4 Functional elements of IL13(EQ)41BBZ-T2A-CD19t_epHIV7 RegulatoryLocation Elements (Nucleotide and Genes Numbers) Comments U5  87-171 5′Unique sequence psi 233-345 Packaging signal RRE  957-1289Rev-responsive element flap 1290-1466 Contains polypurine track sequenceand central termination sequence to facilitate nuclear import ofpre-integration complex EF1p Promoter 1524-2067 EF1-alpha EukaryoticPromoter sequence driving expression of CD19Rop IL13-IgG4 (EQ)-2084-4753 Therapeutic insert 41BB-Zeta-T2A- CD19t WPRE 4790-5390Woodchuck hepatitis virus derived regulatory element to enhance viralRNA transportation delU3 5405-5509 3′ U3 with deletion to generate SINvector R 5510-5590 Repeat sequence within LTR U5 5591-5704 3′ U5sequence in LTR Amp^(R) 6540-7398 Ampicillin-resistance gene CoE1 ori7461-8342 Replication origin of plasmid SV40 ori 8639-8838 Replicationorigin of SV40 CMV promoter 8852-9451 CMV promoter to generate viralgenome RNA R 9507-86  Repeat sequence within LTR

Example 3: Production of Vectors for Transduction of Patient T Cells

For each plasmid (IL13(EQ)BBZ-T2A-CD19t_epHIV7; pCgp; pCMV-G; andpCMV-Rev2), a seed bank is generated, which is used to inoculate thefermenter to produce sufficient quantities of plasmid DNA. The plasmidDNA is tested for identity, sterility and endotoxin prior to its use inproducing lentiviral vector.

Briefly, cells were expanded from the 293T working cell (WCB), which hasbeen tested to confirm sterility and the absence of viral contamination.A vial of 293T cells from the 293T WCB was thawed. Cells were grown andexpanded until sufficient numbers of cells existed to plate anappropriate number of 10 layer cell factories (CFs) for vectorproduction and cell train maintenance. A single train of cells can beused for production.

The lentiviral vector was produced in sub-batches of up to 10 CFs. Twosub-batches can be produced in the same week leading to the productionof approximately 20 L of lentiviral supernatant/week. The materialproduced from all sub-batches were pooled during the downstreamprocessing phase, in order to produce one lot of product. 293T cellswere plated in CFs in 293T medium (DMEM with 10% FBS). Factories wereplaced in a 37° C. incubator and horizontally leveled in order to get aneven distribution of the cells on all the layers of the CF. Two dayslater, cells were transfected with the four lentiviral plasmidsdescribed above using the CaPO4 method, which involves a mixture ofTris:EDTA, 2M CaCl2, 2×HBS, and the four DNA plasmids. Day 3 aftertransfection, the supernatant containing secreted lentiviral vectors wascollected, purified and concentrated. After the supernatant was removedfrom the CFs, End-of-Production Cells were collected from each CF. Cellswere trypsinized from each factory and collected by centrifugation.Cells were resuspended in freezing medium and cryopreserved. These cellswere later used for replication-competent lentivirus (RCL) testing.

To purify and formulate vectors crude supernatant was clarified bymembrane filtration to remove the cell debris. The host cell DNA andresidual plasmid DNA were degraded by endonuclease digestion(Benzonase®). The viral supernatant was clarified of cellular debrisusing a 0.45 μm filter. The clarified supernatant was collected into apre-weighed container into which the Benzonase® is added (finalconcentration 50 U/mL). The endonuclease digestion for residual plasmidDNA and host genomic DNA as performed at 37° C. for 6 h. The initialtangential flow ultrafiltration (TFF) concentration of theendonuclease-treated supernatant was used to remove residual lowmolecular weight components from the crude supernatant, whileconcentrating the virus ˜20 fold. The clarified endonuclease-treatedviral supernatant was circulated through a hollow fiber cartridge with aNMWCO of 500 kD at a flow rate designed to maintain the shear rate at˜4,000 sec-1 or less, while maximizing the flux rate. Diafiltration ofthe nuclease-treated supernatant was initiated during the concentrationprocess to sustain the cartridge performance. An 80% permeatereplacement rate was established, using 4% lactose in PBS as thediafiltration buffer. The viral supernatant was brought to the targetvolume, representing a 20-fold concentration of the crude supernatant,and the diafiltration was continued for 4 additional exchange volumes,with the permeate replacement rate at 100%.

Further concentration of the viral product was accomplished by using ahigh speed centrifugation technique. Each sub-batch of the lentiviruswas pelleted using a Sorvall RC-26 plus centrifuge at 6000 RPM (6,088RCF) at 6° C. for 16-20 h. The viral pellet from each sub-batch was thenreconstituted in a 50 mL volume with 4% lactose in PBS. Thereconstituted pellet in this buffer represents the final formulation forthe virus preparation. The entire vector concentration process resultedin a 200-fold volume reduction, approximately. Following the completionof all of the sub-batches, the material was then placed at −80° C.,while samples from each sub-batch were tested for sterility. Followingconfirmation of sample sterility, the sub-batches were rapidly thawed at37° C. with frequent agitation. The material was then pooled andmanually aliquoted in the Class II Type A/B3 biosafety cabinet in theviral vector suite. A fill configuration of 1 mL of the concentratedlentivirus in sterile USP class 6, externally threaded O-ring cryovialswas used. Center for Applied Technology Development (CATD)'s QualitySystems (QS) at COH released all materials according to the Policies andStandard Operating Procedures for the CBG and in compliance with currentGood Manufacturing Practices (cGMPs).

To ensure the purity of the lentiviral vector preparation, it was testedfor residual host DNA contaminants, and the transfer of residual hostand plasmid DNA. Among other tests, vector identity was evaluated byRT-PCR to ensure that the correct vector is present. All releasecriteria were met for the vector intended for use in this study.

Example 4: Preparation of T Cells Suitable for Use in ACT

T lymphocytes are obtained from a patient by leukopheresis, and theappropriate allogenic or autologous T cell subset, for example, CentralMemory T cells (T_(CM)), are genetically altered to express the CAR,then administered back to the patient by any clinically acceptablemeans, to achieve anti-cancer therapy.

Suitable T_(CM) can be prepared as follows. Apheresis products obtainedfrom consented research participants are ficolled, washed and incubatedovernight. Cells are then depleted of monocyte, regulatory T cell andnaïve T cell populations using GMP grade anti-CD14, anti-CD25 andanti-CD45RA reagents (Miltenyi Biotec) and the CliniMACS™ separationdevice. Following depletion, negative fraction cells are enriched forCD62L+ T_(CM) cells using DREG56-biotin (COH clinical grade) andanti-biotin microbeads (Miltenyi Biotec) on the CliniMACS™ separationdevice.

Following enrichment, T_(CM) cells are formulated in complete X-Vivo15plus 50 IU/mL IL-2 and 0.5 ng/mL IL-15 and transferred to a Teflon cellculture bag, where they are stimulated with Dynal ClinEx™ Vivo CD3/CD28beads. Up to five days after stimulation, cells are transduced withIL13(EQ)BBZ-T2A-CD19t_epHIV7 lentiviral vector at a multiplicity ofinfection (MOI) of 1.0 to 0.3. Cultures are maintained for up to 42 dayswith addition of complete X-Vivo15 and IL-2 and IL-15 cytokine asrequired for cell expansion (keeping cell density between 3×10⁵ and2×10⁶ viable cells/mL, and cytokine supplementation every Monday,Wednesday and Friday of culture). Cells typically expand toapproximately 10⁹ cells under these conditions within 21 days. At theend of the culture period cells are harvested, washed twice andformulated in clinical grade cryopreservation medium (Cryostore CS5,BioLife Solutions).

On the day(s) of T cell infusion, the cryopreserved and released productis thawed, washed and formulated for re-infusion. The cryopreservedvials containing the released cell product are removed from liquidnitrogen storage, thawed, cooled and washed with a PBS/2% human serumalbumin (HSA) Wash Buffer. After centrifugation, the supernatant isremoved and the cells resuspended in a Preservative-Free Normal Saline(PFNS)/2% HSA infusion diluent. Samples are removed for quality controltesting.

Two qualification runs on cells procured from healthy donors wereperformed using the manufacturing platform described above. Eachpreclinical qualification run product was assigned a human donor (HD)number—HD006.5 and HD187.1. Importantly, as shown in Table 5, thesequalification runs expanded >80 fold within 28 days and the expandedcells expressed the IL13(EQ)BBγ/CD19t transgenes.

TABLE 5 Summary of Expression Data from Pre- clinical Qualification RunProduct Cell Product CAR CD19 CD4+ CD8+ Fold Expansion HD006.5 20% 22%24% 76%  84-fold (28 days) Hd187.1 18% 25% 37% 63% 259-fold (28 days)

Example 5: Flow Cytometric Analysis of Surface Transgene and T CellMarker Expression in IL13(EQ)BBγ/CD19t+T_(CM)

The two preclinical qualification run products described in Example 4were used in pre-clinical studies to as described below. FIGS. 6A-Cdepict the results of flow cytometric analysis of surface transgene andT cell marker expression. IL13(EQ)BBγ/CD19t+ T_(CM) HD006.5 and HD187.1were co-stained with anti-IL13-PE and anti-CD8-FITC to detect CD8+ CAR+and CD4+ (i.e., CD8 negative) CAR+ cells (FIG. 6A), or anti-CD19-PE andanti-CD4-FITC to detect CD4+ CD19t+ and CD8+ (i.e., CD4 negative) CAR+cells (FIG. 6B). IL13(EQ)BBγ/CD19t+ T_(CM) HD006.5 and HD187.1 werestained with fluorochrome-conjugated anti-CD3, TCR, CD4, CD8, CD62L andCD28 (grey histograms) or isotype controls (black histograms). (FIG.6C). In each of FIGS. 6A-C, the percentages indicated are based onviable lymphocytes (DAPI negative) stained above isotype.

Example 6: Comparison of CAR T Cell Delivery Route for Treatment ofLarge TS-Initiated PBT Tumors

Described below are studies that compare the route of delivery,intravenous (i.v.) or intracranial (i.c.), on antitumor activity againstinvasive primary PBT lines. In pilot studies (data not shown), it wasunexpectedly observed that i.v. administered IL13(EQ)BBζ+ T_(CM)provided no therapeutic benefit as compared to PBS for the treatment ofsmall (day 5) PBT030-2 EGFP:ffLuc tumors. This is in contrast to therobust therapeutic efficacy observed with i.c. administered CAR+ Tcells. Reasoning that day 5 PBT030-2 tumors may have been too small torecruit therapeutic T cells from the periphery, a comparison was made ofi.v. versus i.c. delivery against larger day 19 PBT030-2 EGFP:ffLuctumors. For these studies, PBT030-2 engrafted mice were treated witheither two i.v. infusions (5×10⁶ CAR+ T_(CM); days 19 and 26) or fouri.c. infusions (1×10⁶ CAR+ T_(CM); days 19, 22, 26 and 29) ofIL13(EQ)BBZ+ T_(CM), or mock T_(CM) (no CAR). Here too no therapeuticbenefit as monitored by Xenogen imaging or Kaplan-Meier survivalanalysis for i.v. administered CAR+ T cells (FIGS. 7A and 7B). Incontrast, potent antitumor activity was observed for i.c. administeredIL13(EQ)BBζ+ T_(CM) (FIGS. 7A-B). Next, brains from a cohort of mice 7days post T cell injection were harvested and evaluated for CD3+ human Tcells by IHC. Surprisingly, for mice treated i.v. with either mockT_(CM) or IL13(EQ)BBζ T_(CM) there were no detectable CD3+ human T cellsin the tumor or in others mouse brain regions where human T cellstypically reside (i.e. the leptomeninges) (FIG. 7C), suggesting adeficit in tumor tropism. This is in contrast to the significant numberof T cells detected in the i.c. treated mice (FIG. 7D).

Tumor derived cytokines, particularly MCP-1/CCL2, are important inrecruiting T cells to the tumor. Thus, PBT030-2 tumor cells wereevaluated and it was found that this line produces high levels ofMCP-1/CCL2 comparable to U251T cells (data not shown), a glioma linepreviously shown to attract i.v. administered effector CD8+ T cells toi.c. engrafted tumors. Malignant gliomas are highly invasive tumors andare often multi-focal in presentation. The studies described aboveestablish that IL13BBZ T_(CM) can eliminate infiltrated tumors such asPBT030-2, and mediate long-term durable antitumor activity. The capacityof intracranially delivered CAR T cells to traffic to multifocal diseasewas also examined. For this study PBT030-2 EGFP:ffLuc TSs were implantedin both the left and right hemispheres (FIG. 8A) and CAR+ T cells wereinjected only at the right tumor site. Encouragingly, for all miceevaluated (n=3) we detected T cells by CD3 IHC 7-days post T cellinfusion both at the site of injection (i.e. right tumor), as wellwithin the tumor on the left hemisphere (FIG. 8B). These findingsprovide evidence that CAR+ T cells are able to traffic to and infiltratetumor foci at distant sites. Similar findings were also observed in asecond tumor model using the U251T glioma cell line (data not shown).

Example 7: Amino Acid Sequence of IL13(EQ)BBζ/CD19t

The complete amino acid sequence of IL13(EQ)BBζ/CD19t is depicted inFIG. 9. The entire sequence (SEQ ID NO:1) includes: a 22 amino acidGMCSF signal peptide (SEQ ID NO:2), a 112 amino acid IL-13 sequence (SEQID NO:3; amino acid substitution E13Y shown in bold); a 229 amino acidIgG4 sequence (SEQ ID NO:4; with amino acid substitutions L235E andN297Q shown in bold); a 22 amino acid CD4 transmembrane sequence (SEQ IDNO:5); a 42 amino acid 4-1BB sequence (SEQ ID NO:6); a 3 amino acid Glylinker; a 112 amino acid CD3ζ sequence (SEQ ID NO:7); a 24 amino acidT2A sequence (SEQ ID NO:8); and a 323 amino acid CD19t sequence (SEQ IDNO:9).

The mature chimeric antigen receptor sequence (SEQ ID NO:10) includes: a112 amino acid IL-13 sequence (SEQ ID NO:3; amino acid substitution E13Yshown in bold); a 229 amino acid IgG4 sequence (SEQ ID NO:4; with aminoacid substitutions L235E and N297Q shown in bold); at 22 amino acid CD4sequence (SEQ ID NO:5); a 42 amino acid 4-1BB sequence (SEQ ID NO:6); a3 amino acid Gly linker; and a 112 amino acid CD3ζ sequence (SEQ IDNO:7). Within this CAR sequence (SEQ ID NO:10) is theIL-13/IgG4/CD4t/41-BB sequence (SEQ ID NO:11), which includes: a 112amino acid IL-13 sequence (SEQ ID NO:3; amino acid substitution E13Yshown in bold); a 229 amino acid IgG4 sequence (SEQ ID NO:4; with aminoacid substitutions L235E and N297Q shown in bold); at 22 amino acid CD4sequence (SEQ ID NO:5); and a 42 amino acid 4-1BB sequence (SEQ IDNO:6). The IL13/IgG4/CD4t/4-1BB sequence (SEQ ID NO:11) can be joined tothe 112 amino acid CD3ζ sequence (SEQ ID NO:7) by a linker such as a GlyGly Gly linker. The CAR sequence (SEQ ID NO:10) can be preceded by a 22amino acid GMCSF signal peptide (SEQ ID NO:2).

FIG. 10 depicts a comparison of the sequences ofIL13(EQ)41BBζ[IL13{EQ}41BBζ T2A-CD19t_epHIV7; pF02630] (SEQ ID NO:12)and CD19Rop_epHIV7 (pJ01683) (SEQ ID NO:13).

Example 8: Amino Acid Sequence of Additional CAR Targeting IL13Rα2

FIGS. 11-18 depict the amino acid sequences of additional CAR directedagainst IL13Rα2. In each case the various domains are labelled exceptfor the GlyGlyGly spacer located between certain intracellular domains.Each includes human IL13 with and Glu to Tyr (SEQ ID NO:3; amino acidsubstitution E13Y shown in highlighted). In the expression vector usedto express these CAR, the amino acid sequence expressed can include a 24amino acid T2A sequence (SEQ ID NO:8); and a 323 amino acid CD19tsequence (SEQ ID NO:9) to permit coordinated expression of a truncatedCD19 sequence on the surface of CAR-expressing cells.

A panel of CAR comprising human IL13(E13Y) domain, a CD28 tm domain, aCD28gg costimulatory domain, a 4-1BB costimulatory domain, and a CD3ζdomain CAR backbone and including either a HL (22 amino acids) spacer, aCD8 hinge (48 amino acids) spacer, IgG4-HL-CH3 (129 amino acids) spaceror a IgG4(EQ) (229 amino acids) spacer were tested for their ability tomediate IL13Rα2-specific killing as evaluated in a 72-hour co-cultureassay. With the exception of HL (22 amino acids) which appeared to havepoor CAR expression in this system, all were active.

Example 9: Structure of Two HER2-CAR

One CAR comprising a HER2 scFv described herein is referred to asHer2scFv-IgG4(L235E, N297Q)-CD28tm-CD28gg-Zeta-T2A-CD19t. This CARincludes a variety of important features including: a scFv targeted toHER2; an IgG4 Fc region that is mutated at two sites within the CH2region (L235E; N297Q) in a manner that reduces binding by Fc receptors(FcRs); a CD28 transmembrane domain, a CD28 co-stimulatory domain, andCD3ζ activation domain. FIG. 20 presents the amino acid sequence of thisCAR, including the sequence of the truncated CD19 sequence used formonitoring CAR expression and the T2A ribosomal skip sequence thatallows the CAR to be produced without fusion of the truncated CD19sequence. As shown in FIG. 21, the immature CAR includes: GMCSFR signalpeptide, HER2 scFv, IgG4 that acts as a spacer, a CD8 transmembranedomain, a 4-IBB co-stimulatory domain that includes a LL to GG sequencealteration, a three Gly sequence, CD3 Zeta stimulatory domain. Thetranscript also encodes a T2A ribosomal sequence and a truncated CD19sequence that are not part of the CAR protein sequence. The mature CARis identical to the immature CAR, but lacks the GMCSF signal peptide.

Example 10: Expression of CAR Targeted to HER2

FIG. 22A is a schematic diagram of two the HER2-specific CAR constructsdepicted in FIG. 20 and FIG. 21. In HER2(EQ)28ζ the scFv is tethered tothe membrane by a modified IgG4 Fc linker (double mutant, L235E; N297Q),containing a CD28 transmembrane domain, an intracellular CD28co-stimulatory domain and a cytolytic CD3ζ domain. The T2A skip sequenceseparates the CAR from a truncated CD19 (CD19t) protein employed forcell tracking. HER2(EQ)BBζ is similar except that the costimulatorydomain is 4-1BB rather than CD28 and the transmembrane domain is a CD8transmembrane domain rather than a CD28 transmembrane domain. Humancentral memory (TCM) cells were transfected with a lentiviral vectorexpressing either HER2(EQ)28ζ or HER2(EQ)BBζ. FIG. 22B depictsrepresentative FACS data of human TCM surface phenotype. FIG. 22Cdepicts the results of assays for CD19 and Protein L expression in TCMtransfected with a lentiviral vector expressing either HER2(EQ)28ζ orHER2(EQ)BBζ. As can be seen from these results, transfection efficiencyas assessed by CD19 expression was similar for both CAR. However,Protein L expression was lower for HER2(EQ)BBζ than for HER2(EQ)28ζsuggesting that the HER2(EQ)BBζ CAR is less stable that the HER2(EQ)BBζ.Analysis of cell expansion (FIG. 22D) shows that neither CAR interfereswith T cell expansion.

Example 11: In Vitro Characterization of HER2 Targeted CAR

A variety of breast cancer cell lines, including, HER2-negative lines(LCL lymphoma, MDA-MB-468, U87 glioma), low-HER2 expressing lines(MDA-MB-361, 231BR) and high-HER2 expressing lines (SKBR3, BT474, BBM1)were used to characterize HER2(EQ)28ζ and HER2(EQ)BBζ. FIG. 23A depictsthe HER2 expression level of each of these lines. Flow cytometry (gatedon CAR+ T cells) was used to characterize CD107a degranulation and IFNγproduction in Mock (untransduced), HER2(EQ)28ζ or HER2(EQ)BBζ CAR Tcells following a 5 hr co-culture with either MDA-MB-361 tumor cells(low HER2 expressing) or BBM1 tumor cells (high HER2 expressing). Theresults of this analysis are presented in FIG. 23B. Similar studies wereconducted with the other breast cancer cells lines, and the results aresummarized in FIG. 23C. Production of IFNγ production by HER2-CAR Tcells following a 24 hr culture with recombinant HER2 protein or tumortargets was measured by ELISA and the results of this analysis are shownin FIG. 23D.

Example 12: In Vitro Anti-Tumor Activity of HER2 Targeted CAR

Flow cytometry was used to assess tumor cell killing following a 72 hco-culture of Mock (untransduced), HER2(EQ)28ζ or HER2(EQ)BBζ CAR Tcells with tumor targets. The results of this analysis are presented inFIG. 24A. PD-1 and LAG-3 induction in total CAR T cells after a 72 hco-culture with HER2-negative MDA-MB-468 or HER2-positive BBM1 cells wasmeasured, and the results of this analysis are presented in FIG. 24B.PD-1 induction in CD8+ CAR T cells following a 72 h co-culture withtumor targets that are HER2-negative (LCL lymphoma, MDA-MB-468, U87glioma), low-HER2 expressing (MDA-MB-361, 231BR) or high-HER2 expressing(SKBR3, BT474, BBM1) was measured, and the results of this analysis arepresented in FIG. 24C. These studies suggest that HER2(EQ)BBζ causeslower PD-1 induction that does HER2(EQ)28ζ. Tumor cell killing withEffector:Tumor (E:T) ratio ranging from 0.25:1 to 2:1 was measured forboth HER2(EQ)28ζ or HER2(EQ)BBζ CAR T cells. The results of thisanalysis are presented in FIG. 24D, which shows that both HER2(EQ)28ζand HER2(EQ)BBζ are effective in tumor cell killing in vitro. CFSEproliferation of HER2-CAR T cells following a 72 h co-culture withMDA-MB-468 or BBM1 cells was measured by flow cytometry. The results ofthis analysis are presented in FIG. 24E, which shows that HER2(EQ)BBζCAR T cells proliferate more than HER2(EQ)28ζ CAR T cells.

Example 13: In Vivo Anti-Tumor Activity of HER2 Targeted CAR

The activity of intratumorally delivered HER2 CAR T cells was assessedin a patient-derived breast-to-brain metastasis model. FIGS. 25A-25C areH&E staining of tumors. Mice were treating by injection directly intothe tumor with Mock (untransduced) or HER2(EQ)BBζ CAR T cells. FIGS.25D-25F depict the results of optical imaging of the tumors and FIGS.25G-251 are Kaplan-Meier survival curves for mice treated locally witheither at day 3, 8 or 14 post tumor injection. These studies show thatHER2(EQ)BBζ CAR T cells have potent anti-tumor efficacy in vivo wheninjected directly into the tumor.

To assess anti-tumor efficacy in human xenograft models ofbreast-to-brain metastasis, BBM1 cells (0.2M) or BT474 (0.15M) wereintracranially injected in NSG mice. At day 8 post tumor injection,HER2(EQ)28ζ or HER2(EQ)BBζ, or Mock (untransduced) T cells (1M) wereinjected intratumorally. BBM1 (FIG. 26A) and BT474 (FIG. 26B) tumorswere monitored by luciferase-based optical imaging. Kaplan Meier curvesare presented in FIG. 26C and FIG. 26D.

A human patient-derived orthotopic xenograft model of breast-to-brainmetastasis was also used to assess HER2(EQ)28ζ and HER2(EQ)BBζ CAR Tcells. FIG. 27A illustrates the region of tumor implantation bystereotactic injection of BBM1 cells (0.2M), and intraventricular T celldelivery. Staining of tumors is depicted in FIG. 27B. At day 14 posttumor injection, HER2(EQ)28ζ, HER2(EQ)BBζ, or Mock (unstransduced) Tcells (0.5M) were injected intratumorally. Tumor growth was monitored byluciferase-based optical imaging. FIG. 27C presents the flux averagesfor each treatment group, and FIG. 27D presents the Kaplan Meiersurvival curve for each treatment group.

Example 14: Comparison of Intracranial and Intratumoral Administrationof T_(CM) Expressing a CAR Targeted to IL13Rα2

Two different intracranial (ic) delivery routes, intratumoral (ict) andintraventricular (icy) were assessed in a murine model of glioblastomafor in vivo safety, trafficking and efficacy of CAR T cells generatedfrom T_(CM)-enriched cell lines that were transduced with theIL13(EQ)BBZ-T2A-CD19t_epHIV7 lentiviral vector and expanded in vitro asproposed for the clinical treatment of glioblastoma (GBM). In vivosafety and functional potency of these cells administered either ict oricv was examined in immunodeficient NSG mice using the IL13Rα2+ primarylow-passage GBM tumor sphere line PBT030-2, which has been engineered toexpress the firefly luciferase (ffLuc) reporter gene.

T_(CM) cell lines that had been enriched from PBMC byCliniMACS™/AutoMACS selection were lenti-transduced withIL13(EQ)BBZ-T2A-CD19t_epHIV7 lentivirus, expanded and then cryopreservedusing methods similar to that described above. Freshly thawed CAR Tcells administered either ict or icv were then evaluated for potentialtoxicity, their ability to traffic to multifocal GBM tumors and theirpotency in controlling the in vivo growth of ic engrafted IL13Rα2+ GBMline PBT030-2 cells. To assess general toxicity, mice were observeddaily for overall health, including body weight and alertness. Tumorburden, as measured by Xenogen imaging, was examined; andimmunohistochemistry (IHC) to detect T cell recruitment/infiltration ofthe tumors was also performed on a subset of mice.

Male NSG mice (10-12 weeks old) were stereotactically injected ic with1×10⁵ ffLuc+ PBT030-2 cells in both the right and left contralateralhemispheres on day 0 and allowed to engraft for 6 days. Mice were thengrouped based on tumor size as determined by Xenogen imaging for equaltumor size distributions per group. Groups of mice were then leftuntreated (n=4), or treated either ict (right hemisphere, n=8) or icv(n=8) with 1×10⁶ CAR+IL13(EQ)BBζ/CD19t+ T_(CM) (FIG. 28). PBT030-2 tumorgrowth was monitored over time by Xenogen imaging and quantification offfLuc flux (photons/sec). At different time points, mice from each groupwere euthanized, their brains harvested, embedded in paraffin andimmunohistochemistry (IHC) was performed to evaluate the presence ofhuman CD3-expressing cells (i.e., human T cells). Specifically, threemice were euthanized from each CAR T cell treated group one week afterthe T cell administration (Day 13 of the experiment), and thus thesemice were not included in the Xenogen imaging analysis of FIG. 29; andthen two mice in each of the groups of mice were euthanized two weeksafter the T cell administration (Day 21 of the experiment).

While this was not a survival study, and thus mice were all euthanizedat specific time points to evaluate T cell trafficking in the brains(described below), the mice were monitored daily for any obvious signsof distress or general toxicity. Mice treated with either the ict or icvregimen did not exhibit any weight loss, and were bright, alert andreactive throughout the experiment. Thus, regardless of the route of Tcell administration, there were no signs of any therapy-associatedadverse effects.

As shown in FIGS. 29A-C, ict delivery of IL13(EQ)BBζ/CD19t+T_(CM)exhibited robust anti-tumor activity against the PBT030-2 tumors asexpected. However, icv delivery of IL13(EQ)BBζ/CD19t+ T_(CM) appeared toprovide greater therapeutic benefit against is engrafted PBT030-2 tumorsthan that observed with ict administration, especially against the tumorlesion in the contralateral (left) hemisphere.

To determine if route of administration affected the ability of T cellsto migrate to the tumor site, IHC analysis for CD3+ T cells wasperformed on the brains of mice from each group at one and two weeksafter the T cell administration. As shown in FIG. 30, human CD3+ T cellswere found at both the left and right tumor sites in mice that hadreceived either ict or icv administered T cells. These data arerepresentative of 3 mice in each group at one week, and 2 mice in eachgroup at two weeks post T cell administration.

This study demonstrates that both intratumoral and intraventricularadministration of T cells were well-tolerated in this NSG mouse model.Furthermore, in vivo multi-focal anti-tumor efficacy and IHC detectionof T cells at the tumor sites can be observed with both ict and icydelivery of T_(CM) qualification run cells that had been transduced withthe IL13(EQ)BBZ-T2A-CD19t_epHIV7 vector. This study further suggeststhat icv delivered T cells may have greater efficacy than ict deliveredT cells.

Example 15: Phase 1 Clinical Trial Evaluating IL-13Rα2 CAR T Cells forTreatment of Glioblastoma

This example describes the initial findings of a clinical trialevaluating the safety, feasibility and bioactivity of weeklyintracranial infusions of autologous IL13BBζ Tcm in patients withrecurrent IL13Rα2+ GBM. As described in greater detail below, Enrolledpatients undergo leukapheresis to collect autologous PBMC and,concurrent with IL13BBζ+ Tcm manufacturing, tumor biopsy or resection isperformed, with placement of a reservoir/catheter device. Followingbaseline MR and PET imaging and recovery from surgery, patients aretreated on a 4-week therapeutic regimen, consisting of 3-weeklyintracranial infusions of IL13BBζ+ Tcm followed by one rest week fortoxicity and disease assessment. The results to date for this first lowdose cohort of three resection patients, suggest that local delivery ofIL13BBζ Tcm post surgical resection is safe and well-tolerated with nograde 3 or higher toxicities attributed to the therapy observed, andimportantly, demonstrate early evidence for antitumor activity followingCAR T cell administration. For all patients in which a sample wasavailable, CAR T cells were detected in the tumor cyst fluid or cerebralspinal fluid (CSF) by flow cytometry for a minimum of 7 days posttreatment. One patient of particular interest presented with a recurrentmultifocal GBM, including one metastatic site in the spine and extensiveleptomeningeal disease. This patient was initially treated per protocolwith six local infusions of IL13BBζ Tcm into the resection cavity of thelargest recurrent tumor focus in the posterior temporal-occipitalregion. Encouragingly, this CAR T cell injection site remained stablewithout evidence of disease recurrence for over 7-weeks, while otherdisease foci distant from the CAR T cell injection site continued toprogress. This patient was then treated on a compassionate use protocolwith five weekly intraventricular infusions of IL13BBζ Tcm without anyother therapeutic interventions. One week following the finalintraventricular CAR T cell infusion, all intracranial and spinal tumorshad regressed with most decreasing more than 75% by volume, and thispatient remained clinically stable four months following the start ofCAR T cell treatment.

The CAR, IL13(EQ)BBζ, used in this study is described above. Thesequence of the immature CAR, including the CD19t marker is depicted inFIG. 9. The entire immature sequence (SEQ ID NO:1) includes: a 22 aminoacid GMCSF signal peptide (SEQ ID NO:2), a 112 amino acid IL-13 sequence(SEQ ID NO:3; amino acid substitution E13Y shown in bold); a 229 aminoacid IgG4 sequence (SEQ ID NO:4; with amino acid substitutions L235E andN297Q shown in bold); a 22 amino acid CD4 transmembrane sequence (SEQ IDNO:5); a 42 amino acid 4-1BB sequence (SEQ ID NO:6); a 3 amino acid Glylinker; a 112 amino acid CD3ζ sequence (SEQ ID NO:7); a 24 amino acidT2A sequence (SEQ ID NO:8); and a 323 amino acid CD19t sequence (SEQ IDNO:9).

Autologous cells from each patient was used to prepare CD8+ CD4+ T_(CM)cells which were then transfected with a lentiviral vector, describedabove, expressing IL13(EQ)BBζ. Briefly, T_(CM) were enriched fromperipheral blood mononuclear cells (PBMC) using the CliniMACS® device toimmunomagnetically select for CD45RO+/CD62L+ T_(CM). These cells wereactivated with anti-CD3/CD28 Dynal beads, transduced with a SINlentiviral vector that directs the expression of the IL13(EQ)BBζ CAR.The activated/genetically modified IL13(EQ)BBζ/CD19t+ T_(CM) cells wereexpanded in vitro with IL-2/IL-15 and then cryopreserved.

The treatment of two patients, both suffering from relapsed/refractoryGBM is described below. Intracavity administration of CAR T cells wasperformed manually over about 5-10 minutes through a Rickham catheterfollowed by up to 1.0 mL preservative-free normal-saline (PFNS) flushdelivered by convection enhanced delivery (CED) at 0.5 ml/hour.Intraventricular administration of CAR T cells was performed manuallyover approximately 5-10 minutes through a Rickham catheter placed intothe lateral ventricle. This was followed by up to 0.5 mLpreservative-free normal-saline (PFNS) flush delivered via a manual pushtechnique over 5-10 minutes. The PFNS flush is meant to clear theadministration line and push remaining CAR T cells through the catheter.

The time course of CAR T cell preparation and treatment is depicted inFIG. 31. Concurrent with the manufacturing process, researchparticipants underwent resection of their tumor(s) followed by placementof a Rickham catheter and baseline imaging.

Patient UPN097 underwent tumor resection and was treated in Cycle 1 with2×10⁶ cells and in Cycle 2 with 10×10⁶ cells. In both Cycle 1 and Cycle2 the cells were administered to the cavity left by resection. After thesecond cycle Patient UPN097 was taken off the study due to rapid tumorprogression.

Patient UPN109 was treated in Cycle 1 with 2×10⁶ cells and in Cycles 2and 3 with 10×10⁶ cells. After a rest period, Patient UPN109 was treatedin Cycles 4, 5 and 6 with 10×10⁶ cells. In Cycles 1-6 the cells wereadministered into. In Cycle 7 the patient was treated with 2×10⁶ cells.In Cycles 8 and 9 the patient was treated with 10×10⁶ cells. In Cycles7-10 the administration was intraventricular.

FIG. 32A presents analysis of CAR T cell persistence, as monitored byCD19. This analysis shows good T cell persistence 8 days after the Cycle2. FIG. 32B shows decreased presence of GBM cells as monitored byIL13Rα2 expression on cells.

FIG. 33A and FIG. 33B depict imaging results from Patient UPN097 in theregion of the catheter used for intratumoral administration. In FIG. 33Aone can see that few CD3+ or CD8+ T cells are present pretreatment. FIG.33B, which is a sample at Day 16 post-treatment taken from the leftfrontal tumor cavity wall shows a large area of necrotic tumor andsignificant presence of CD3+ and CD8+ cells.

As shown in FIGS. 34A-D, there was an increase in IFN-gamma (a Th1cytokine) over the two Cycles of treatment while levels of IL-13 (a Th2cytokine did not change significantly (FIG. 34A and FIG. 34B). IL-6, atumor related cytokine, decreased during Cycle 1 and remained at thelower level during Cycle 2 (FIG. 34C). IL-8, another tumor relatedcytokine, decreased during Cycle 1, but increased towards its pre-Cycle1 level during Cycle 2 (FIG. 34D).

Patient UPN109 presented with a recurrent multifocal GBM, including onemetastatic site in the spine and extensive leptomeningeal disease. Asdescribed above, this patient was treated with six local infusions ofIL13BBζ Tcm into the resection cavity of the largest recurrent tumorfocus. While the CAR T cell injection site remained stable withoutevidence of disease recurrence for over 7-weeks, other disease focidistant from the CAR T cell injection site continued to progress (datanot shown). This patient then received five weekly intraventricularinfusions of IL13BBζ Tcm, as described above. FIGS. 35A-B presents MMand/or PET images of transverse brain section (FIG. 35A) and saggitalbrain section (FIG. 35B). FIG. 35C presents transverse (top) and frontal(bottom) sections of the spine before (left) and one week after (right)completion of intraventricular therapy, with tumor lesion sitesindicated by red arrows in each image.

Example 16: Case Report on Intraventricular Administration

A 50 year old male was initially diagnosed with a low-grade brain tumorin the right temporal lobe after presenting with grand mal seizures.After four months of monitoring, this right temporal tumor displayedincreased enhancement by MRI, and the patient underwent tumor resectionwhich confirmed a diagnosis of WHO grade IV glioma (GBM). The patientthen received standard-of-care adjuvant proton radiation to a total doseof 59.4 cobalt Gy equivalent with concurrent temozolomide (140 mgdaily), followed by 4-cycles of temozolomide with concomitant use of theNovocure device (NovoTTF-100A) for three months. Six months after theprimary tumor resection, PET and MRI images showed evidence of diseaseprogression.

The patient was then treated autologous IL13Rα2-targeted CAR T cells(FIG. 36) following confirmation of IL13Rα2-expression in the primaryright temporal lobe tumor by IHC, with an H score of 100 (FIG. 37).Peripheral blood mononuclear cells (PBMC) were then collected byleukapheresis followed by enrichment of CD4+ CD8+ TCM via a two-stepdepletion selection procedure as previously described. During IL13BBζTCM manufacturing, the patient was treated on a separate clinicalprotocol evaluating a fibroblastic growth factor inhibitor(NCT01975701), and progressed through therapy with symptoms ofheadaches, confusion and disorientation increasing. Additionally, thepatient was tapered off steroids prior to T cell injections.

Ten months post primary tumor resection, the patient underwent anothersurgical resection for three of five identified progressing GBM lesions(FIG. 38), including the largest lesion in the right posteriortemporal-occipital region (T1) where the reservoir/catheter device wasplaced, and two lesions in the right frontal lobe (T2, T3). Twoadditional tumors in the left temporal area were not surgically removed(T4, T5). Six days post-surgical resection, the patient received two4-week treatment regimens, each consisting of three weekly intracavitary(ICT) infusions of IL13BBζ TCM followed by a week for toxicityevaluation and disease assessment. This patient was treated startingwith a low dose of 2×10⁶ CAR+ T cells followed by five infusions of10×10⁶ CAR+ T cells (FIG. 39). Following these six ICT infusions, undera compassionate use protocol, a second catheter was placed in the rightlateral ventricle, allowing the patient to receive an additional fiveintracerebroventricular (ICV) treatment cycles of IL13BBζ TCM, againstarting at a low dose of 2×10⁶ CAR T cells followed by four infusionsof 10×10⁶ CAR T cells (FIG. 40).

Due to limited therapeutic product only five ICV infusion cycles werefeasible. Overall, the patient received 11 cell infusions for a totaldose of 94×10⁶ CAR+ T cells. The treatment course encompassed 15-weeks,with evaluation weeks for toxicity and disease assessment (i.e., MM andPET imaging) taking place after every third cycle, and after the finaltwo ICV infusions. The patient received no other therapeuticinterventions during this CAR T cell treatment course, and findings upto the 190 day evaluation period, encompassing the 11 infusions cycles,is reported here. Subsequently, a second IL13BBζ TCM product wasmanufactured and beginning on day 192 this patient has continued toreceive ICV infusions of this second manufactured product approximatelyevery 3 weeks.

Example 17: Study Design

These studies, including the compassionate use protocol, were approvedby an institutional review board, and the patient provided writteninformed consent. Eligibility included prior histologically-confirmeddiagnosis of an IL13Rα2+ grade IV glioma that is now recurrent, age>18years with a Karnofsky performance status (KPS)>60, adequatecardiopulmonary function, and a survival expectation>4 weeks. Thepatient must have completed initial radiation therapy at least 12 weeksprior to enrollment, and must not have any other active malignancies,infections or intercurrent illness or be receiving other investigationalagents or require more than 2 mg TID (3×/day) of Dexamethasone during Tcell therapy.

This patient was initially treated under our ongoing phase I study(NCT02208362) to evaluate the safety and feasibility of weeklyintracranial infusions of autologous IL13Rα2-targeted CAR T cells(IL13BBζ TCM) in patients diagnosed with recurrent/refractory IL13Rα2+high-grade glioma (WHO Grades III and IV). This is a two arm study withT cells administered either directly into the tumor (stratum1=intratumoral) or into the tumor resection cavity (stratum2=intracavitary). After completing the six intracavitary (ICT) infusioncycles, this patient was then treated under a separate compassionate useprotocol allowing for intracerebroventricular (ICV) delivery of IL13BBζTCM.

Example 18: Cell Product Manufacture and Infusion

The lentiviral vector encoding the 4-1BB costimulatory, IL13Rα2-targetedCAR, IL13BBζ, is detailed herein. Briefly, the codon optimized CARsequence contains a membrane-tethered human IL-13 ligand mutated at asingle site (E13Y) to reduce potential binding to IL13Rα1, a human IgG4Fc spacer containing two mutations (L235E; N297Q) that prevent Fcreceptor-mediated recognition, a human CD4 transmembrane domain, a humancostimulatory 4-1BB cytoplasmic signaling domain, and a human CD3ζcytoplasmic signaling domain. A T2A ribosome skip sequence thenseparates this IL13BBζ CAR sequence from a truncated human CD19 sequence(CD19t), an inert, nonimmunogenic cell surface marker.

For IL13BBζ TCM manufacturing, on the day of leukapheresis, PBMC wereisolated by density gradient centrifugation over Ficoll-Paque (GEHealthcare) followed by two washes in PBS/EDTA. PBMC were then washedonce in PBS, resuspended in X Vivo15 media (Bio Whittaker) containing10% fetal calf serum (FCS) (Hyclone), transferred to a 300 cc transferbag, and stored on a 3-D rotator overnight at room temperature (RT). Thefollowing day, 5×109 PBMC were incubated in a 300 cc transfer bag withclinical grade anti-CD14 (1.25 mL), anti-CD25 (2.5 mL) and anti-CD45RA(2.5 mL) microbeads (Miltenyi Biotec) for 30 minutes at RT in X Vivo15containing 10% FCS. CD14+, CD25+, and CD45RA+ cells were thenimmediately depleted using the CliniMACS™ depletion mode according tothe manufacturer's instructions (Miltenyi Biotec). After centrifugation,the unlabeled negative fraction of cells was resuspended in CliniMACS™PBS/EDTA buffer (Miltenyi Biotec) containing 0.5% human serum albumin(HSA) (CSL Behring) and then labeled with clinical gradebiotinylated-DREG56 mAb (COHNMC CBG) at 0.6 mL for 30 minutes at RT. Thecells were then washed and resuspended in a final volume of 100 mLCliniMACS™ PBS/EDTA containing 0.5% HSA and transferred into a new 300cc transfer bag. After 30 minutes incubation with 1.25 mL anti-biotinmicrobeads (Miltenyi Biotec), the CD62L+ fraction (TCM) was purifiedwith positive selection on CliniMACS™ according to the manufacturer'sinstructions, and resuspended in X Vivo15 containing 10% FCS.

Within 2 hours of enrichment, 26.9×10⁶ TCM were stimulated with GMPDynabeads® Human T expander CD3/CD28 (Invitrogen) at a 1:3 ratio (Tcell:bead), and transduced with clinical grade IL13BBζ-T2A-CD19t_epHIV7at an MOI of 0.3 in 5.5 mL X Vivo15 containing 10% FCS with 5 μg/mLprotamine sulfate (APP Pharmaceutical), 50 U/mL rhIL-2 and 0.5 ng/mLrhIL-15 in a 32 Vuelife tissue culture bag (AFC) that was placed at ahorizontal position on a culture rack at 37° C., 5% CO2. Cultures werethen maintained with addition of X-Vivo15 10% FCS as required to keepcell density between 4×10⁵ and 2×10⁶ viable cells/mL, with cytokinesupplementation (final concentration of 50 U/mL rhIL-2 and 0.5 ng/mLrhIL-15) every Monday, Wednesday and Friday of culture. Based on culturevolume, T cells were transferred to 730 Vuelife bags (AFC). Seven daysafter the lentiviral transduction, the CD3/CD28 Dynabeads were removedusing the Dynal ClinEx Vivo Magnetic Particle Concentrator bag magnet,and bead-free T cells were drained into a new 730 Vuelife bag. Cultureswere propagated until approximately 4.53×10⁸ cells were generated asdetermined by Guava PCA, at which time cultures were harvested, washedin Isolyte (Braun) with 2% HSA, then resuspended in Cryostor CS5(BioLife Solutions) at approximately 1.3×10⁷ cells/mL forcryopreservation using a Mr. Frosty (Nalgene) and a portable controlledrate freezer system (Custom Biogenics). Quality control tests includedviability, potency (CD19t expression), Identity (CD3 expression),transgene copy number (WPRE qPCR), replication competent virus testing(VSV-G qPCR and formal RCL testing at the University of Indiana),residual bead count, and sterility.

As noted above, the T2A ribosome skip sequence 12 then separates thisIL13BBζ sequence from CD19t, an inert, nonimmunogenic cell surfacemarker marking cell transduction (FIG. 39A). This T2A linkage results inthe coordinate expression of both IL13BBζ and CD19t from a singletranscript.

Manufacturing methods for the immunomagenetic enrichment ofCD62L+CD45RA-CD4+CD8+ central memory T cells (TCM), lentiviraltransduction and ex vivo expansion are also detailed herein. Theend-of-process (EOP) cyropreserved IL13BBζ TCM product underwent qualitycontrol release testing as per the clinical protocol. For each infusion,T cells were thawed, washed and reformulated into a final volume of 0.5mL in pharmaceutical preservative-free normal saline (PFNS) with 2%human serum albumin (HSA). Cells were manually injected into the Rickhamreservoir using a 21 gauge butterfly needle to deliver a 0.5 mL volumeover 5-10 minutes, followed by up to 1 mL PFNS flush delivered byconvection enhanced delivery (CED) at 0.5 mL per hour.

Example 19: Clinical Imaging

The post-gadolinium T1 weighted MRI sequences of the brain and spinewere acquired on a Siemens Viro 3 Tesla scanner. Lesions were measuredon axial T1 MPR weighted images obtained after the administration ofMultihance. Imaging with 18-F-fluorodeoxyglucose (18-F-FDG) wasperformed using a GE Discovery DST HP60 PET-CT scanner (70 cm axialfield of view, slice thickness 3.75 mm). Maximal standardized uptakevalues (SUVs) were obtained utilizing Vital Images Vitrea version 6.7.2software. Regions of contrast-enhancing tumor foci were outlined by aradiologist for measurements of largest tumor area (mm2) and tumorvolumes (cm3) were computed.

Cryopreserved cell banks of quality control released autologous IL13BBζTCM were thawed and reformulated for infusion by washing twice withphosphate buffered saline (PBS) with 2% HSA and resuspending inpharmaceutical preservative-free normal saline with 2% HSA. Delivery ofthe therapeutic CAR T cells into either the glioma resection cavity(ICT) or the lateral ventricle (ICV) was achieved using a Holter™Rickham Ventriculostomy Reservoir (Codman), with a ventricular catheter(Integra Pudenz), and a stylet. For ICT delivery, the reservoir/cathetersystem was inserted at the time of tumor resection, and the tip of thecatheter was partially embedded into the resection wall in order toallow for cell delivery both into the cavity and into the peritumoralbrain tissue. Post-operative imaging (CT and MRI) were obtained toconfirm catheter position and extent of tumor resection.

Example 20: IL13BBζ TCM Display a Central Memory-Like T Cell Phenotype

Enriched TCM (36×10⁶) were ex vivo stimulated, lentivirally transducedand expanded to yield 638×10⁶ total cells in 17 days. The final T cellproduct (CD3+ and TCR+) consisted of CD4 (74%) and CD8 (16%) T cellsubsets and expressed IL13BBζ and CD19t with gene modified co-stainingfor both cell surface proteins (FIG. 39A). The CAR T cell productexhibited a central memory T cell phenotype, expressing CD45RO (97%),CD62L (57%), CCR7 (28%), CD28 (97%) and CD27 (59%) (FIG. 39A). Theproduct also expressed of some markers of exhaustion, including TIM-3(65%) and LAG-3 (49%), but not significant levels of PD-1 and KLRG1(FIG. 39A).

Example 21: Safety and Tolerability of Repetitive IntracranialInfustions of IL13BBζ TCM

The patient was treated with weekly infusions of IL13BBζ TCMadministered via a reservoir/catheter device through two differentintracranial delivery routes, that being intracavitary (ICT) deliveryfollowing tumor resection (cycles 1-6) and intracerebroventricular (ICV)delivery into the cerebral spinal fluid (CSF) (cycles 7-11). The 11intracranial infusions, at a maximum cell dose of 10×10⁶ CAR+ T cells,were well-tolerated with no grade 3 or higher toxicity (NCI CommonToxicity Criteria) with possible or higher attribution to the therapyobserved. Mild events noted following CAR T cell infusions include.

TABLE 6 SAFETY AND TOLERABILITY Maximum Cumulative Delivery T-cellT-cell T-cell Adverse Event Route Doses Dose Dose (Grade 1-2)* ICT 6 10⁷5.2 × 10⁷ Chills Fatigue Fever Lymphopenia Myalgia Dizziness HeadacheSeizure ICV 5 10⁷ 4.2 × 10⁷ Chills Fatigue Fever Myalgia Headache ShortOlfactory Aura Seizure Anxiety Hypertension Sinus Tachycardia *Onlyevents with possible or higher attribution to the T cell administrationare reported; all occurred once and were Grade 1-2 according to the NCICommon Toxicity Criteria, with no events of Grade 3 or higher observed.

Example 22: Clinical Response

At the time of treatment, the patient's tumor displayed characteristicsof a highly aggressive recurrent GBM with poor prognostic features. Thisincluded evidence of recurrence from primary diagnosis within six monthsfollowing standard-of-care therapy, the presentation of multifocal tumorlesions, including spinal lesions and extensive leptomeningeal disease(FIG. 38), histological features of a dedifferentiated GBM, and a hightumor proliferation rate with over 60% of the cells staining positivefor Ki67 (FIG. 37B). Tumor expression of IL13Rα2, as evaluated by IHC onFFPE of resected tumor tissue, was similar between the primary andrecurrent tumors with an H score of 100 and 80, respectively (FIG. 37).Intratumoral IL13Rα2 expression for the recurrent tumor washeterogeneous, with 10% of the cells showing high staining intensity(2-3+), 60% showing low expression (1+), and 30% of the cells notstaining above background (0+). Of potential interest, the highestlevels of IL13Rα2 expression were often observed in tumor regions ofpseudopalisading necrosis (FIG. 37A), an expression pattern noted forother GBM tumors.

Following enrollment on the clinical protocol, this patient underwentsurgical resection for three of the five recurrent lesions (FIG. 38),and the reservoir/catheter device was place in the resected cavity ofthe largest recurrent foci (T1) in the right posteriortemporal-occipital area. This patient was initially treated per protocolwith six weekly intracavitary (ICT) infusions of CAR+ T cells (2×10⁶cycle 1; 10×10⁶ cycles 2-6) (FIG. 1B), and during this time period thetemporal-occipital tumor lesion (T1) remained stable for over 45-dayspost-surgery without evidence of progressive disease (FIG. 39C). MMrevealed, however, that other non-resected tumor foci in the lefttemporal lobes (T4 and T5), as well as a new recurrent lesion adjacentto tumor 3 (T6) and a lesion in the olfactory groove (T7) continued toprogress over this same time period (FIG. 39C). Additionally, metastaticlesions in the spine, including one large tumor (270 mm²) and more thanone small tumor foci (<1 cm²) were also detected. These results, whilemixed, were encouraging, in that they suggested the IL13BBζ T_(CM) mayhave prevented disease recurrence at the resected posteriortemporal-occipital area, however, they also suggested that local ICTdelivery was not sufficient to effectively control tumor progression atdistant locations away from the infusion site.

Based on these findings and supported by preclinical studies showingthat ICV delivery of CAR T cells can traffic to multifocal GBM in NSGmouse models (data not shown), this patient was enrolled on acompassionate use protocol and treated with five weekly ICV infusions ofIL13BBζ T_(CM) without any other therapeutic interventions (2×10⁶ cycle7; 10×10⁶ cycles 8-11) (FIG. 40A). One week after three ICV infusions(cycle 9; day 133) all tumor lesions showed dramatic regression, andfollowing the final ICV infusion (cycle 11; day 156), most intracranialand spinal tumors had regressed more than 70% by both maximum area andvolume measurements (FIG. 40B-E, Table 7 below, and FIG. 41). Follow-upMR and PET imaging six weeks after the last ICV infusion (day 190),during which the patient received no other therapeutic intervention,showed continued disease regression, with all tumors decreasing ≧78% byboth maximum area and volume measurements (FIG. 40B-E, Table 7 below,and FIG. 41). At this 190 day time point, it was not possible todifferentiate between residual radiographic evidence of disease versusinflammation, scaring and/or dural enhancement. ICV delivery of IL13BBζT_(CM) elicited almost complete elimination of all metastatic tumors inthe spine, with 97% reduction in the maximum area for the largest lesionand only one small tumor foci visible out of the more than ten presentprior to ICV treatment. Over the time course of ICV treatment, andcoinciding with tumor regression, the patient was able to reduce systemdexamethosome from 2 mg bid to 0.5 mg qd. This patient remainsclinically stable and has returned to normal life and work activities,thus supporting the durability of this CAR T cell-mediated antitumorresponse. These results demonstrate that treatment with IL13BBζ T_(CM)mediated a near complete response based on the stringent RANO criteria.

TABLE 7 MRI Evaluation of Non-Resected Lesions (Volume in cm³, Area inmm²) Anatom- Post Op Post Post Post Op Post Post Tu- ical Pre Op i.c.t.Cycles 1-3 Cycles 4-6 i.c.v. Cycles 7-9 Cycles 10-11 Max % mor LocationD50 D51 D77 D86 D101 D108 D133 D156 D190 Decrease 4 Left 0.2 cm³ 0.3 cm³0.5 cm³ ND 0.8 cm³ 1.4 cm ³ 0.3 cm³ 0.1 cm³ 0.1 cm ³ Vol: 93% temporal,65 mm² 98 mm² 112 mm² 168 mm² 224 mm ² 80 mm² 49 mm² 28 mm ² Area: 88%pterion 5 Left 0 cm³ 0 cm³ 0.1 cm³ ND 0.3 cm³ 0.7 cm ³ 0.1 cm³ 0 cm³ 0cm ³ Vol: 100% temporal, 20 mm² 20 mm² 36 mm² 54 mm² 126 mm ² 33 mm² 11mm² 7 mm ² Area: 94% apex  6* Right NA NA 0.5 cm³ ND 1 cm³ 1.7 cm³ 1.8cm ³ 1.4 cm³ 0.4 cm ³ Vol: 78% frontal 0 mm² 0 mm² 42 mm² 176 mm² 187mm² 300 mm ² 143 mm² 64 mm ² Area: 79% lobe  7* Olfactory NA 0.1 cm³ 0.4cm³ ND 1.4 cm³ 2.5 cm ³ 1.9 cm³ 1.3 cm³ 0.3 cm ³ Vol: 88% groove 27 mm²18 mm² 60 mm² 171 mm² 360 mm ² 312 mm² 98 mm² 40 mm ² Area: 89% 8 SpinalND ND ND 270 mm ² ND ND 35 mm² 18 mm² 8 mm ² Area: 97% *new lesionarising during Cycles 1-6 Bold, values compared for Maximum % DecreaseNA, no lesion could be identified 0, lesion might be visuallyidentified, but value was below that of analysis software parameters ND,imaging was not done

Example 23: CAR T Cell Persistence and CNS Inflammatory Response

To elucidate immunological changes associated with antitumor responsesobserved following the ICV infusion of IL3BBζ T_(CM) (Cycles 6-11), CSFwas evaluated for cell infiltrates, CAR+ T cell persistence, andcytokine levels. Immediately following each ICV infusion (i.e., day 1-2of cycles 6-11), cell numbers per mL of CSF increased 7.0±3.6 fold ascompared to pre-infusion levels (day 0 of each cycle), and increased153±128 fold as compared to pre-ICV (C7D0) levels (FIG. 42). Total cellnumbers in the CSF typically decreased over the 7-day treatment cycle.As evaluated for C9D2, the cell infiltrates included a large proportionof CD3+ T cells, both endogenous and CAR-expressing, as well asCD14+CD11b+ HLA-DR+ mature myeloid populations (FIG. 42A). Only rareCD19+ B cells and CD11b+CD15+ granulocytes were detected (FIG. 42A).Consistent with flow cytometry data, CSF cytopathology on C11D1 reportedthe presence of reactive lymphocytes, monocytes, and macrophages.

CAR-T persistence was also monitored over the ICV treatment course. Dueto low cell recovery in the CSF for cycles 7 and 8, analysis focused onevaluating cycle 9 and time points immediately following cycles 10 and11. Importantly, CAR+ T cells were detected at all-time points evaluated(FIG. 42B), including C9D0 which corresponded to 7-days post cycle 8,thus demonstrating persistence of the therapeutic cells for at least 7to 8 days post-infusion. However, CAR T numbers in the CSF post infusiondecreased at the later cycles (C10D1 and C11D1) when tumor burden hadalso significantly decreased (FIG. 40B). To note, significant expansionof the CAR T cells in the CSF over cycle 9 was not detected, with CAR+cell numbers increasing 1.6-fold 2 days later (C9D2) from pre-infusion(C9D0) and then decreasing 2.3-fold by day 8 (C9D8).

The presence of immune cells, including CAR+ T cells, following eachinfusion corresponded to significant elevations of cytokine levels inthe CSF. The measured levels and calculated fold-change over baselinefor the 30-cytokines measured is presented in Tables 9 and 10 below.Notably, 11 cytokines increased more than 10-fold from pre-ICV baseline(C7D0) immediately following IL3BBζ T_(CM) infusions, includingcytokines IFNγ, TNF, IL-2, IL-10, IL-5, IL-6, and IL-8 and chemokinesCXCL9/MIG, CXCL10/IP-10, and CCR2/MCP-1 and soluble cytokine receptorIL-1Rα (FIG. 3C). Seven other cytokines showed greater than a 5-foldincrease from baseline (C7D0), including G-CSF, IL-12, IL2-R, IL-4,IL-7, and MIP-lb. The inflammatory cytokines that exhibited the highestfold increase immediately following IL3BBζ T_(CM) infusion as comparedto pre-ICV (C7D0) was IL-2 (>90-fold for C9D2) and the IFN-γ induciblechemokines CXCL9 and CXCL10 (>40-fold for C8D1, C9D2, C10D1 and C11D2).These cytokines returned to near baseline levels within 7-days betweentreatment cycles. Cytokines that did not show significant increasefollowing CAR T cell infusions include IL-13, RANTES and VEGF (Tables 9and 10).

TABLE 9 UPN 109 CSF Cytokine Analysis (pg/mL), ICV Cycles 7 through 11.Cytokine C7D0 C7D2 C8D0 C8D1 C9D0 C9D2 C9D8 C10D0 C10D1 C11D0 C11D1C11D44 EGF OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< 10.0Eotaxin *2.1 *2.4 *2.4 6.2 3.0 3.8 *2.4 *2.4 6.3 *2.4 6.1 *2.6 FGF  7.18.0 8.8 14.1 *4.0 *3.1 *4.7 *6.3 12.2 7.3 13.6 11.5 G-CSF *25.1  68.6*43.7 232.6 103.9 137.5 64.9 *23.3 245.6 *13.8 248.9 39.9 GM-CSF *2.0*2.4 *2.5 *8.7 *2.5 *4.2 *1.4 *1.3 *3.2 *1.3 *2.7 OOR< HGF 74.4 113.1127.7 253.9 162.3 250.8 145.8  110.9 213.6 125.2 241.8 81.0 IFN-α 45.556.8 42.0 109.7 59.5 66.1 35.6 17.9 90.0 24.8 74.5 OOR< IFN-γ *8.2 *7.0*3.8 140.8 16.8 32.1 *5.0 *1.8 69.5 *4.0 42.8 *1.0 IL-10 *4.4 *6.0 *2.174.6 *20.7 70.4 *16.0  *3.5 147.1 *6.9 167.5 OOR< IL-12 16.7 23.5 24.792.4 41.5 82.7 62.4 35.5 57.0 42.6 85.7 12.7 IL-13 *15.8  *15.3 *13.129.9 *15.9 18.1 *4.8 OOR< 22.7 OOR< 18.8 OOR< IL-15 OOR< OOR< OOR< OOR<OOR< OOR< OOR< OOR< OOR< OOR< OOR< *7.1 IL-17 *2.4 *2.8 *0.9 *9.2 *5.4*5.1 *2.3 *1.2 *8.5 *1.0 *8.1 OOR< IL-1Rα *50.1  *35.7 *56.9 405.9 238.6699.3 358.0  605.2 1113.0 *53.4 1141.9 259.9  IL-1β *5.0 10.1 *6.2 22.1*3.0 12.9 *6.7 *6.7 15.6 *8.0 17.0  *4.69 IL-2 OOR< *4.2 *0.8 55.4 *1.0*2.7 OOR< *0.6 10.8 *0.6 *5.5 OOR< IL-2R 43.8 81.0 51.2 223.7 89.6 243.167.7 *13.1 219.3 *18.3 241.5 54.2 IL-4 *2.5 *3.8 *2.9 *17.0 *5.5 *8.3*3.9 *2.3 *13.8 *1.3 *10.5 OOR< IL-5 OOR< *1.3 *0.5 14.7 *2.6 9.1 *1.0OOR< 7.9 OOR< 7.7 OOR< IL-6 56.5 78.4 40.9 1062.5 106.5 318.4 47.0 33.2688.5 31.4 857.3 23.2 IL-7 OOR< 6.3 OOR< 42.7 20.0 19.9 OOR< 6.3 23.4OOR< 22.0 28.0 IL-8 226.2  231.0 253.4 4904.6 827.4 1591.0 677.8  283.21023.9 84.4 794.9 66.0 IP-10 161.4  766.7 307.3 6213.7 916.7 59779.1510.1  156.9 393430.8 345.3 305579.5 79.2 MCP-1 1660.6  1752.3 1280.818439.9 4437.4 1939.1 791.9  1598.9 10868.4 420.0 3157.4 888.7  MIG 82.9302.1 179.1 4500.5 1360.6 3621.2 1342.1  380.7 3423.0 288.2 3823.6 29.3MIP-1α 22.0 28.0 20.7 68.1 31.9 50.8 19.7 *14.8 68.6 *14.6 64.4 *8.8MIP-1β 26.3 33.8 26.1 213.8 49.7 106.1 24.2 16.8 126.8 22.3 52.6 13.6RANTES *15.5  OOR< OOR< 41.7 25.7 OOR< OOR< OOR< 68.5 *1.0 *12.5 OOR<TNF-α OOR< OOR< OOR< 19.9 *1.6 *6.3 OOR< OOR< 11.0 OOR< *5.1 OOR< VEGF17.0 21.8 16.7 90.2 25.5 38.6 10.9 7.8 65.5 OOR< 70.0 14.1 OCR<, Out ofRange (below) *Value extrapolated beyond standard range

TABLE 10 UPN 109 CSF Cytokine Fold Change Analysis, ICV Cycles 7 through11. Cytokine C7D0 C7D2 C8D0 C8D1 C9D0 C9D2 C9D8 C10D0 C10D1 C11D0 C11D1C11D44 EGF 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Eotaxin 1.01.1 1.1 3.0 1.4 1.8 1.1 1.1 3.0 1.1 2.9 1.2 FGF 1.0 1.1 1.2 2.0 0.6 0.40.7 0.9 1.7 1.0 1.9 1.6 G-CSF 1.0 2.7 1.7 9.3 4.1 5.5 2.6 0.9 9.8 0.59.9 1.6 GM-CSF 1.0 1.2 1.3 4.4 1.3 2.1 0.7 0.7 1.6 0.7 1.4 0.7 HGF 1.01.5 1.7 3.4 2.2 3.4 2.0 1.5 2.9 1.7 3.3 1.1 IFN-α 1.0 1.2 0.9 2.4 1.31.5 0.8 0.4 2.0 0.5 1.6 0.5 IFN-γ* 1.0 0.9 0.5 17.2 2.0 3.9 0.6 0.2 8.50.5 5.2 0.1 IL-10* 1.0 1.4 0.5 17.0 4.7 16.0 3.6 0.8 33.4 1.6 38.1 0.5IL-12 1.0 1.4 1.5 5.5 2.5 5.0 3.7 2.1 3.4 2.6 5.1 0.8 IL-13 1.0 1.0 0.81.9 1.0 1.1 0.3 0.3 1.4 0.3 1.2 0.3 IL-15 1.0 1.0 1.0 1.0 1.0 1.0 1.01.0 1.0 1.0 1.0 1.0 IL-17 1.0 1.2 0.4 3.8 2.3 2.1 1.0 0.5 3.5 0.4 3.40.4 IL-1Rα* 1.0 0.7 1.1 8.1 4.8 14.0 7.1 12.1 22.2 1.1 22.8 5.2 IL-1β1.0 2.0 1.2 4.4 0.6 2.6 1.3 1.3 3.1 1.6 3.4 0.9 IL-2* 1.0 7.0 1.3 92.31.7 4.5 1.0 1.0 18.0 1.0 9.2 1.0 IL-2R 1.0 1.8 1.2 5.1 2.0 5.6 1.5 0.35.0 0.4 5.5 1.2 IL-4 1.0 1.5 1.2 6.8 2.2 3.3 1.6 0.9 5.5 0.5 4.2 0.5IL-5* 1.0 2.6 1.0 29.4 5.2 18.2 2.0 1.0 15.8 1.0 15.4 1.0 IL-6* 1.0 1.40.7 18.8 1.9 5.6 0.8 0.6 12.2 0.6 15.2 0.4 IL-7 1.0 1.0 1.0 6.8 3.2 3.21.0 1.0 3.7 1.0 3.5 4.4 IL-8* 1.0 1.0 1.1 21.7 3.7 7.0 3.0 1.3 4.5 0.43.5 0.3 IP-10* 1.0 4.8 1.9 38.5 5.7 370.4 3.2 1.0 2437.6 2.1 1893.3 0.5MCP-1* 1.0 1.1 0.8 11.1 2.7 1.2 0.5 1.0 6.5 0.3 1.9 0.5 MIG* 1.0 3.6 2.254.3 16.4 43.7 16.2 4.6 41.3 3.5 46.1 0.4 MIP-1α 1.0 1.3 0.9 3.1 1.5 2.30.9 0.7 3.1 0.7 2.9 0.4 MIP-1β 1.0 1.3 1.0 8.1 1.9 4.0 0.9 0.6 4.8 0.82.0 0.5 RANTES 1.0 0.1 0.1 2.7 1.7 0.1 0.1 0.1 4.4 0.1 0.8 0.1 TNF-α*1.0 1.0 1.0 12.4 1.0 3.9 1.0 1.0 6.9 1.0 3.2 1.0 VEGF 1.0 1.3 1.0 5.31.5 2.3 0.6 0.5 3.9 0.5 4.1 0.8 Bold values, ‘OOR<’ value from Table 10was replaced with the lowest measurable value for that cytokine to allowfor fold change calculation. *Cytokines in which a >10 fold increase wasobserved at least once

These immunological changes in the CSF were local, as no significantchanges in cytokine levels (Table 11), and no detectable CAR+ T cells byqPCR and flow cytometry (data not shown) in the peripheral blood wereobserved. The changes in the CSF could not be compared to changes in theresected cavity of tumor lesion 1 (T1) due to the inability to obtaincyst fluid from the cavity during the ICT treatment course.

TABLE 11 UPN 109 Serum Cytokine Analysis (pg/mL), ICV Cycles 7 through11. Cytokine C7D0 C7D2 C7D4 C8D0 C8D1 C8D4 C9D0 C9D2 C10D0 C10D1 C10D3C11D0 C11D1 C11D2 EGF 148.1 166.7 171.9 168.8 132.2  118.3 105.9 73.8154.8 158.9 121.9 114.8 152.2 151.8 Eotaxin 110.1 116.8 112.6 101.2 83.7133.8 152.1 156.4 172.1 167.9 143.2 147.3 197.7 168.3 FGF *5.3 8.3 6.8OOR< OOR< OOR< OOR< 14.7 17.4 22.7 20.2 14.8 15.3 15.9 G-CSF 211.6 236.7284.5 229.3 208.2  208.2 210.8 230.1 216.7 334.3 221.8 282.9 207.4 241.6GM-CSF *2.0 *2.1 *2.4 *1.9 *1.6 *2.0 *1.9 *1.7 *2.0 *3.1 *2.3 *1.9 *1.8*1.7 HGF 471.5 596.1 611.6 420.1 403.0  508.1 362.4 400.0 385.9 502.5456.6 395.3 476.1 451.6 IFN-α 43.9 47.4 49.9 43.8 42.2 47.1 43.9 40.041.1 64.5 43.1 50.4 43.3 43.4 IFN-γ 53.0 52.5 56.4 52.2 52.1 55.5 52.244.8 45.6 58.5 47.0 54.8 49.7 50.7 IL-10 *2.9 *3.9 *3.4 *0.8 OOR< *2.4*1.0 *3.3 *2.6 *9.4 *3.5 *1.8 *1.9 *0.6 IL-12 211.7 192.3 195.0 187.2182.3  190.9 192.8 223.3 227.9 241.6 254.4 220.2 240.5 219.1 IL-13 21.127.4 30.7 23.1 36.8 31.0 28.0 24.2 22.3 38.3 31.4 32.2 25.8 34.8 IL-15OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR<IL-17 *3.5 *4.1 *5.2 OOR< *1.8 *0.7 *0.8 *3.3 *4.6 *11.5 *5.2 *7.3 *4.4*4.4 IL-1Rα 112.7 145.3 *68.2 *94.3 *73.8  *64.5 *58.2 *64.9 101.1 133.896.3 *68.7 107.9 105.8 IL-1β *2.1 *4.6 *4.9 *1.0 OOR< OOR< OOR< 11.315.9 30.6 18.2 12.7 14.3 14.7 IL-2 *0.1 *0.9 *1.3 *0.3 OOR< *0.2 *0.3*0.4 *0.9 *5.2 *1.2 *1.8 *0.9 *1.0 IL-2R 372.0 391.2 438.5 352.6 273.9 272.7 241.9 304.9 312.2 363.8 314.8 338.0 296.4 314.5 IL-4 *8.9 *11.5*13.5 *9.0 *10.4  *10.5 *10.1 *10.2 *8.7 *20.9 *11.1 *13.8 *9.7 *10.8IL-5 *1.6 *2.0 *3.2 *0.2 *1.5 *1.1 *0.8 OOR< OOR< 5.1 *0.2 *2.2 OOR<OOR< IL-6 OOR< *1.6 *0.4 OOR< *0.7 OOR< OOR< *2.5 *2.6 7.1 *4.2 *2.9*3.9 *3.0 IL-7 OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR<OOR< OOR< OOR< IL-8 49.4 130.2 88.4 96.4 56.3 17.1 43.6 *9.4 32.8 93.1112.1 18.3 30.1 59.0 IP-10 33.9 23.5 17.2 11.7  9.0 11.5 15.0 12.7 17.433.6 16.3 15.3 23.2 18.9 MCP-1 459.6 610.9 475.9 426.2 414.8  561.0944.8 538.5 848.2 1074.3 703.0 954.9 950.0 826.3 MIG 141.3 108.1 50.68.0 OOR< 10.0 28.5 34.0 41.2 79.1 42.6 47.3 42.2 44.2 MIP-1α 58.4 58.462.2 51.1 49.2 53.4 53.1 47.1 55.8 81.2 57.7 64.6 52.7 54.1 MIP-1β 103.393.4 92.0 78.1 64.4 76.8 83.1 57.3 84.5 157.1 86.3 90.0 87.6 90.0 RANTES11127.1 11965.0 14328.5 10584.5 12610.1   12415.5 12937.9 9221.7 8567.410428.1 8886.3 11117.8 9782.5 9771.1 TNF-α *1.2 *2.1 *4.5 *2.3 *2.6 *2.1*2.3 OOR< OOR< 7.2 OOR< *2.1 OOR< OOR< VEGF OOR< OOR< OOR< OOR< OOR<OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR< OOR<, Out of Range (below)*Value extrapolated beyond standard range

Example 24: Patient Sample Processing and Analysis

Tumor resection material was collected through the COH department ofPathology according to the clinical protocol.

IL13Rα2 immunohistochemistry (IHC) was performed on 5 μm-sections offormalin-fixed paraffin-embedded specimens as previously described, andKi67 IHC was similarly performed with the exception of antigen retrievalby heating @ pH 8.0, and incubation with a 1:75 dilution of anti-K167(Dako Corp). IL-13Rα2 immunoreactivity was scored by a clinicalneuropathologist and quantified based on the percentage of tumor cellsexhibiting weak (1+), moderate (2+), or strong (3+) intensity ofcytoplasmic and golgi-like staining. The H score is obtained by theformula: (3×percentage of strongly staining cells)+(2×percentage ofmoderately staining cells)+percentage of weakly staining cells, giving arange of 0 to 300. The H score can be translated into the intensityscoring system described in the enrollment criteria as follows: 0representing negative (H score 0), 1+ low (H score 1-100), 2+ moderate(H score 101-200) and 3+ high (H score 201-300). The criteria forinclusion was at least 20% of the cells scoring 1+ staining intensity(>20%, 1+), representing an H score of 20. Appropriate positive(testicular) and negative (prostate) controls were employed for IL-13Rα2IHC staining. A “+” sign reflects the presence of membranous staining.This test has been performed at the Department of Pathology, City ofHope National Medical Center and is regarded as investigational forresearch. This Laboratory is certified under the Clinical LaboratoryImprovement Amendments of 1988 (CLIA) as qualified to perform highcomplexity clinical laboratory testing.

Peripheral blood samples were collected in vacutainer tubes±EDTA.Samples with EDTA were ficolled immediately upon receipt and peripheralblood mononuclear cells (PBMC) were frozen in Crystor CS5 at −80° C. andthen transferred to liquid nitrogen for long term storage. Sampleswithout EDTA were allowed to coagulate for 2-3 hours at roomtemperature; serum was collected by centrifugation, aliquoted in singleuse 100-200 μl aliquots and stored at −80° C. Cerebral spinal fluid(CSF) was collected from the ICV reservoir in a 3 cc syringe, spun down,and supernatants were aliquoted and stored at −80° C. The CSF cells wereresuspended in HBSS−/− (Corning CellGro) with 2% FCS and sodium azidefor immediate flow cytometric analysis, with the remaining cellsresuspended and frozen in Cryostor CS4 at −80° C. and then transferredto liquid nitrogen for long term storage

Cell surface phenotyping of immune cells was performed by flow cytometryusing fluorochrome conjugated antibodies specific for CD3, CD4, CD11b,CD14, CD19, CD27, CD28, CD62L, CD45RA, CD45RO, IL-13, TCR-α/β (BDBiosciences), KLRG1, CD15 (BioLegend), HLA-DR, PD1 (eBiosciences), CD8(Fisher Scientific), LAG-3 (Lifespan Biosciences), CCR7, or TIM-3 (R&DSystems), and their respective isotype controls.

Research participant serum and CSF samples were analyzed by cytokinebead array. Assays were performed using the Human Cytokine 30-Plex Panelkit (Invitrogen) and a FLEXMAP 3D® (Luminex).

1. A method of treating a patient diagnosed with a malignancy of thecentral nervous system comprising introducing into the cerebrospinalfluid (CSF) of the patient a composition comprising an effective amountof T cells.
 2. The method of claim 1 wherein the T cells are autologousor allogenic T cells.
 3. The method of claim 1 wherein the T cells havebeen manipulated ex vivo by one or more of: expansion, fractionation ortransfection with a recombinant nucleic acid molecule.
 4. The method ofclaim 3 wherein the T cells comprise cells that have been transfectedwith a recombinant nucleic acid molecule encoding a polypeptide thatbinds to a tumor cell antigen.
 5. The method of claim 4 wherein thepolypeptide is a chimeric antigen receptor.
 6. The method of claim 1wherein the composition is administered intraventricularly
 7. The methodof claim 1 wherein the composition is administered to the central canalof the spinal cord.
 8. The method of claim 6 wherein the administrationis to the left ventrical or the right ventrical.
 9. The method of claim1 wherein the composition comprises at least 1×10⁶ cells.
 10. The methodof claim 1 wherein a composition comprising T cells is administered atleast two times.
 11. The method of claim 10 wherein the wherein theadministrations differ in the total number of T cells administered. 12.The method of claim 10 wherein the administrations escalate in dose. 13.The method of claim 10 wherein the administration de-escalate in dose.14. The method of claim 1 wherein the T cells comprise CAR T cellsexpressing a chimeric antigen receptor.
 15. The method of claim 1wherein the T cells comprise autologous tumor infiltrating lymphocytes.16. The method of claim 1 wherein the T cells comprise TCR-engineered Tcells.
 17. The method of claim 1 wherein the malignancy is a diffuse,infiltrating tumor.
 18. The method of claim 1 wherein the malignancy isa primary brain tumor.
 19. The method of claim 1 wherein one or moretumor foci decrease in size by at least 25%.
 20. The method of claim 1wherein the malignancy arose from a primary cancer selected from: breastcancer, lung cancer, head and neck cancer, and melanoma.
 21. The methodof claim 1 wherein the method is performed after tumor resection. 22.The method of claim 1 further comprising intratumoral administration ofa composition comprising T cells.
 23. The method of claim 1 wherein themalignancy is secondary brain tumor.
 24. The method of claim 1 furthercomprising intratumoral administration of a composition comprisingtherapeutic T cells expressing a chimeric antigen receptor that binds aprotein expressed on the surface of glioblastoma cells.
 25. The methodof claim 24 wherein the patient has previously undergone resection of atumor lesion. 26.-124. (canceled)